Measuring Receptor Homodimerization

ABSTRACT

The invention provides methods and kits for detecting and/or measuring receptor homodimers on a cell surface membrane. In one aspect, the methods employ pairs of probes comprising binding compounds and a cleaving probe, such that at least one binding compound binds specifically to the same epitope of a membrane-bound analyte as the cleaving probe. The binding compound includes one or more molecular tags attached through a cleavable linkage, and the cleaving probe includes a cleavage-inducing moiety that can cleave the linkage when within a defined proximity thereto. Binding of the two probes to a homodimer of a cell surface molecules results in release of molecular tags from the binding compounds, providing a measure of formation of the homodimeric complex.

This patent application is a continuation of U.S. patent applicationSer. No. 10/946,816 filed on 22 Sep. 2004, now U.S. Pat. No. 7,402,398which is a continuation-in-part of U.S. patent application Ser. No.10/623,057 filed 17 Jul. 2003, now U.S. Pat. No. 7,105,308 and claimspriority from U.S. Provisional Applications Ser. No. 60/508,034 filed 1Oct. 2003; Ser. No. 60/566,352 filed 28 Apr. 2004; and Ser. No.60/577,256 filed 3 Jun. 2004, all of which are incorporated herein byreference in their entireties, and from which priority is claimed.

FIELD OF THE INVENTION

The present invention relates to methods for measuring oligomerizationof cell surface molecules, particularly homodimers of cell surfacemembrane receptors.

BACKGROUND OF THE INVENTION

The interactions of cell surface membrane components play crucial rolesin transmitting extracellular signals to a cell in normal physiology,and in disease conditions. In particular, many types of cell surfacereceptors undergo dimerization or oligomerization in connection with thetransduction of an extracellular event or signal, e.g. ligand-receptorbinding, into a cellular response, such as proliferation, increased ordecreased gene expression, or the like, e.g. George et al, NatureReviews Drug Discovery, 1: 808-820 (2002); Mellado et al, Ann. Rev.Immunol., 19: 397-421 (2001); Schlessinger, Cell, 103: 211-225 (2000);Yarden, Eur. J. Cancer, 37: S3-S8 (2001). The role of such signaltransduction events in diseases, such as cancer, has been the object ofintense research and has led to the development of several new drugs anddrug candidates, e.g. Herbst and Shin, Cancer, 94: 1593-1611 (2002);Yarden and Sliwkowski, Nature Reviews Molecular Cell Biology, 2: 127-137(2001).

A wide variety of techniques have been used to study dimerization andoligomerization of cell surface receptors, includingimmunoprecipitation, chemical cross-linking, bioluminescence resonanceenergy transfer (BRET), fluorescence resonance energy transfer (FRET),and the like, e.g. Price et al, Methods in Molecular Biology, 218:255-267 (2003); McVey et al, J. Biol. Chem., 17: 14092-14099 (2001);Salim et al, J. Biol. Chem., 277: 15482-15485 (2002); Angers et al,Proc. Natl. Acad. Sci., 97: 3684-3689 (2000). Unfortunately, despite theimportance of receptor dimerization and oligomerization in signaltransduction processes, the techniques for measuring such interactionsare difficult to apply, lack flexibility, and lack sensitivity. The lackof a convenient and sensitive technique for analyzing theoligomerization of cell surface molecules has greatly increased thedifficulty of developing new therapeutics or diagnostic methods based onsuch phenomena.

In view of the above, the availability of a convenient, sensitive, andcost effective technique for detecting or measuring the dimerization oroligomerization of cell surface analytes would advance the art in manyfields where such measurements are becoming increasingly important,including life science research, medical research and diagnostics, drugdiscovery, and the like.

SUMMARY OF THE INVENTION

The invention provides methods of detecting and/or measuring oligomersof membrane-bound molecules, and especially, homodimers andhomo-oligomers of cell membrane receptors. In one aspect, the method ofthe invention uses at least two reagents that are specific for membersof a dimer or oligomer: one member, referred to herein as a cleavingprobe, has a cleavage-inducing moiety that may be induced to cleavesusceptible bonds within its immediate proximity; and the other member,referred to herein as a binding compound, has one or more molecular tagsattach by linkages that are cleavable by the cleavage-inducing moiety.In accordance with the method, whenever a homodimer or homo-oligomerforms, a fraction of such complexes, especially homodimers, will havespecifically bound at least one cleaving probe and at least one bindingcompound. Under such conditions, the cleavable linkages of the bindingcompounds are brought within the effective cleaving proximity of thecleavage-inducing moieties so that molecular tags can be released. Thereleased molecular tags are then separated from the reaction mixture andquantified to provide a measure of homodimerization orhomo-oligomerization.

In another aspect, the method of the invention may comprising thefollowing steps:

(a) providing a binding compound specific for a membrane-associatedanalyte forming a homodimer, the binding compound having one or moremolecular tags each attached thereto by a cleavable linkage, the one ormore molecular tags each having a separation characteristic;(b) providing a cleaving probe specific for the membrane-associatedanalyte, the cleaving probe having a cleavage-inducing moiety with aneffective proximity, and the cleaving probe and the binding compoundbeing selected such that only one of either the cleaving probe or thebinding composition can specifically bind to the samemembrane-associated analyte at a time;(c) combining the cleaving probe, the binding compound, and the cellmembrane such that the cleaving probe and the binding compoundspecifically bind to membrane-associated analytes and such thatcleavable linkages of the binding compound are within the effectiveproximity of the cleavage-inducing moiety whenever a homodimer ispresent and the cleaving probe and the binding compound specificallybind to different membrane-associated analytes thereof, so thatmolecular tags are released; and (d) separating and identifying thereleased molecular tags to determine the presence or absence or theamount of homodimer in the cell membrane.

In another aspect the method of the invention comprises the followingsteps:

(a) providing one or more binding compounds specific for differentantigenic determinants of a homodimer, each binding compound having oneor more molecular tags each attached thereto by a cleavable linkage, andthe molecular tags of different binding compounds having differentseparation characteristics; (b) providing a cleaving probe specific foran antigenic determinant of the homodimer the same as at least oneantigenic determinant that the one or more binding compounds arespecific for, the cleaving probe having a cleavage-inducing moiety withan effective proximity; (c) mixing the cleaving probe, the one or morebinding compounds, and the cell membrane such that the cleaving probeand the one or more binding compounds specifically bind to theirrespective antigenic determinants and the cleavable linkages of the oneor more binding compounds are within the effective proximity of thecleavage-inducing moiety whenever a homodimer is present and thecleaving probe and at least one binding compound specifically bind todifferent antigenic determinants thereof, so that molecular tags arereleased; and(d) separating and identifying the released molecular tags to determinethe presence or absence or the amount of homodimer in the cell membrane.

In another aspect, the invention includes kits for carrying out themethods of the invention. Such kits comprise at least one cleaving probeand one or more binding compounds having appropriate specificities forthe homodimers to be detected or measured. In one embodiment, cleavingprobes and at least one binding compound of the kit comprise the sameantibody binding composition. In another embodiment, such kits aredesign for the detection of receptor tyrosine kinase homodimers. Inanother embodiment, such kits are designed for the detection of GPCRhomodimers or EGFR homodimers.

In one aspect, the method of the invention uses at least two reagentsthat are specific for different members of a dimer or oligomer: onemember, referred to herein as a cleaving probe, has a cleavage-inducingmoiety that may be induced to cleave susceptible bonds within itsimmediate proximity; and the other member, referred to herein as abinding compound, has one or more molecular tags attach by linkages thatare cleavable by the cleavage-inducing moiety. In accordance with themethod, whenever such different members form a dimer or oligomer, thecleavable linkages are brought within the effective cleaving proximityof the cleavage-inducing moiety so that molecular tag can be released.The molecular tags are then separated from the reaction mixture andquantified to provide a measure of dimerization or oligomerization.

In another aspect, the method of the invention comprises the followingsteps: providing a cleaving probe specific for a first receptor type ofa plurality of receptor types, the cleaving probe having acleavage-inducing moiety with an effective proximity; providing one ormore binding compounds each specific for a different second receptortype of the plurality, each binding compound having one or moremolecular tags each attached thereto by a cleavable linkage, and themolecular tags of different binding compounds having differentseparation characteristics; mixing the cleaving probe, the one or morebinding compounds, and a cell membrane containing the first and secondreceptor types such that the cleaving probe and the one or more bindingcompounds specifically bind to their respective receptors and thecleavable linkages of the one or more binding compounds are within theeffective proximity of the cleavage-inducing moiety so that moleculartags are released; and separating and identifying the released moleculartags to determine the presence or absence or the amount ofoligomerization of the receptor types in the cell membrane.

In another aspect, the invention provides a method of detecting dimersof membrane-associated analytes in a cell membrane, the methodcomprising the steps of: providing a binding compound specific for afirst membrane-associated analyte of a dimer, the dimer comprising thefirst membrane-associated analyte and a second membrane-bound analyte,and the binding compound having one or more molecular tags each attachedthereto by a cleavable linkage, the one or more molecular tags eachhaving a separation characteristic; providing a cleaving probe specificfor the second membrane-bound analyte, the cleaving probe having acleavage-inducing moiety with an effective proximity; mixing thecleaving probe, the binding compound, and the cell membrane such thatthe cleaving probe specifically binds to the first membrane-associatedanalyte and the binding compound specifically binds to the secondmembrane-associated analyte and such that cleavable linkages of thebinding compound are within the effective proximity of thecleavage-inducing moiety so that molecular tags are released; andseparating and identifying the released molecular tags to determine thepresence or absence or the amount of dimer in the cell membrane.

In another aspect, the invention provides a method for profiling thefrequencies of dimers among a plurality of receptor types on thesurfaces of cells.

The present invention provides a method of detecting or measuring thedimerization or oligomerization of membrane-associated analytes,especially homodimers thereof, that has several advantages over currenttechniques including, but not limited to, (1) the detection and/ormeasurement of molecular tags that are separated from an assay mixtureprovide greatly reduced background and a significant gain insensitivity; and (2) the use of molecular tags that are speciallydesigned for ease of separation and detection thereby providingconvenient multiplexing capability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F illustrate diagrammatically the use of releasable moleculartags to measure receptor dimer populations.

FIGS. 1G-1H illustrate diagrammatically the use of releasable moleculartags to measure cell surface receptor complexes in fixed tissuespecimens.

FIGS. 2A-2D illustrate diagrammatically methods for attaching moleculartags to antibodies.

FIG. 3 illustrates the attachment of photosensitizers to antibodies.

FIGS. 4A-4E illustrate data from assays on SKBR-3 and BT-20 cell lysatesfor receptor heterodimers using a method of the invention.

FIGS. 5A-5C illustrate data from assays for receptor heterodimers onhuman normal and tumor breast tissue samples using a method of theinvention.

FIGS. 6A and 6B illustrate data from assays of the invention fordetecting homodimers and phosphorylation of Her1 in lysates of BT-20cells.

FIG. 7 shows data from assays of the invention that show Her2 homodimerpopulations on MCF-7 and SKBR-3 cell lines.

FIGS. 8A-8B show data from assays of the invention that detectheterodimers of Her1 and Her3 on cells in response to increasingconcentrations of heregulin (HRG).

FIGS. 9A and 9B show data on the increases in the numbers of Her1-Her3heterodimers on 22Rv1 and A549 cells, respectively, with increasingconcentrations of epidermal growth factor (EGF).

FIGS. 10A-10C show data on the expression of heterodimers of IGF-1R andvarious Her receptors in frozen samples from human breast tissue.

FIGS. 11A-11G illustrate the assay design and experimental results fordetecting a PI3 kinase-Her3 receptor activation complex.

DEFINITIONS

“Antibody” means an immunoglobulin that specifically binds to, and isthereby defined as complementary with, a particular spatial and polarorganization of another molecule. The antibody can be monoclonal orpolyclonal and can be prepared by techniques that are well known in theart such as immunization of a host and collection of sera (polyclonal)or by preparing continuous hybrid cell lines and collecting the secretedprotein (monoclonal), or by cloning and expressing nucleotide sequencesor mutagenized versions thereof coding at least for the amino acidsequences required for specific binding of natural antibodies.Antibodies may include a complete immunoglobulin or fragment thereof,which immunoglobulins include the various classes and isotypes, such asIgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereofmay include Fab, Fv and F(ab′)2, Fab′, and the like. In addition,aggregates, polymers, and conjugates of immunoglobulins or theirfragments can be used where appropriate so long as binding affinity fora particular polypeptide is maintained. Guidance in the production andselection of antibodies for use in immunoassays, including such assaysemploying releasable molecular tag (as described below) can be found inreadily available texts and manuals, e.g. Harlow and Lane, Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1988);Howard and Bethell, Basic Methods in Antibody Production andCharacterization (CRC Press, 2001); Wild, editor, The ImmunoassayHandbook (Stockton Press, New York, 1994), and the like.

“Antibody binding composition” means a molecule or a complex ofmolecules that comprises one or more antibodies, or fragments thereof,and derives its binding specificity from such antibody or antibodyfragment. Antibody binding compositions include, but are not limited to,(i) antibody pairs in which a first antibody binds specifically to atarget molecule and a second antibody binds specifically to a constantregion of the first antibody; a biotinylated antibody that bindsspecifically to a target molecule and a streptavidin protein, whichprotein is derivatized with moieties such as molecular tags orphotosensitizers, or the like, via a biotin moiety; (ii) antibodiesspecific for a target molecule and conjugated to a polymer, such asdextran, which, in turn, is derivatized with moieties such as moleculartags or photosensitizers, either directly by covalent bonds orindirectly via streptavidin-biotin linkages; (iii) antibodies specificfor a target molecule and conjugated to a bead, or microbead, or othersolid phase support, which, in turn, is derivatized either directly orindirectly with moieties such as molecular tags or photosensitizers, orpolymers containing the latter.

“Antigenic determinant,” or “epitope” means a site on the surface of amolecule, usually a protein, to which a single antibody molecule binds;generally a protein has several or many different antigenic determinantsand reacts with antibodies of many different specificities. A preferredantigenic determinant is a phosphorylation site of a protein.

“Binding moiety” means any molecule to which molecular tags can bedirectly or indirectly attached that is capable of specifically bindingto an analyte. Binding moieties include, but are not limited to,antibodies, antibody binding compositions, peptides, proteins, nucleicacids, and organic molecules having a molecular weight of up to 1000daltons and consisting of atoms selected from the group consisting ofhydrogen, carbon, oxygen, nitrogen, sulfur, and phosphorus. Preferably,binding moieties are antibodies or antibody binding compositions.

“Capillary-sized” in reference to a separation column means a capillarytube or channel in a plate or microfluidics device, where the diameteror largest dimension of the separation column is between about 25-500microns, allowing efficient heat dissipation throughout the separationmedium, with consequently low thermal convection within the medium.

“Chromatography” or “chromatographic separation” as used herein means orrefers to a method of analysis in which the flow of a mobile phase,usually a liquid, containing a mixture of compounds, e.g. moleculartags, promotes the separation of such compounds based on one or morephysical or chemical properties by a differential distribution betweenthe mobile phase and a stationary phase, usually a solid. The one ormore physical characteristics that form the basis for chromatographicseparation of analytes, such as molecular tags, include but are notlimited to molecular weight, shape, solubility, pKa, hydrophobicity,charge, polarity, and the like. In one aspect, as used herein, “highpressure (or performance) liquid chromatography” (“HPLC”) refers to aliquid phase chromatographic separation that (i) employs a rigidcylindrical separation column having a length of up to 300 mm and aninside diameter of up to 5 mm, (ii) has a solid phase comprising rigidspherical particles (e.g. silica, alumina, or the like) having the samediameter of up to 5 μm packed into the separation column, (iii) takesplace at a temperature in the range of from 35° C. to 80° C. and atcolumn pressure up to 150 bars, and (iv) employs a flow rate in therange of from 1 μL/min to 4 mL/min. Preferably, solid phase particlesfor use in HPLC are further characterized in (i) having a narrow sizedistribution about the mean particle diameter, with substantially allparticle diameters being within 10% of the mean, (ii) having the samepore size in the range of from 70 to 300 angstroms, (iii) having asurface area in the range of from 50 to 250 m²/g, and (iv) having abonding phase density (i.e. the number of retention ligands per unitarea) in the range of from 1 to 5 per nm². Exemplary reversed phasechromatography media for separating molecular tags include particles,e.g. silica or alumina, having bonded to their surfaces retentionligands, such as phenyl groups, cyano groups, or aliphatic groupsselected from the group including C₈ through C₁₈. Chromatography inreference to the invention includes “capillary electrochromatography”(“CEC”), and related techniques. CEC is a liquid phase chromatographictechnique in which fluid is driven by electroosmotic flow through acapillary-sized column, e.g. with inside diameters in the range of from30 to 100 μm. CEC is disclosed in Svec, Adv. Biochem. Eng. Biotechnol.76: 1-47 (2002); Vanhoenacker et al, Electrophoresis, 22: 4064-4103(2001); and like references. CEC column may use the same solid phasematerials as used in conventional reverse phase HPLC and additionallymay use so-called “monolithic” non-particular packings. In some forms ofCEC, pressure as well as electroosmosis drives an analyte-containingsolvent through a column.

“Complex” as used herein means an assemblage or aggregate of moleculesin direct or indirect contact with one another. In one aspect,“contact,” or more particularly, “direct contact” in reference to acomplex of molecules, or in reference to specificity or specificbinding, means two or more molecules are close enough so that attractivenoncovalent interactions, such as Van der Waal forces, hydrogen bonding,ionic and hydrophobic interactions, and the like, dominate theinteraction of the molecules. In such an aspect, a complex of moleculesis stable in that under assay conditions the complex isthermodynamically more favorable than a non-aggregated, ornon-complexed, state of its component molecules. As used herein,“complex” usually refers to a stable aggregate of two or more proteins,and is equivalently referred to as a “protein-protein complex.” Mosttypically, a “complex” refers to a stable aggregate of two proteins. Asused herein, an “intracellular complex” or “intracellularprotein-protein complex,” refers to a complex of proteins normally foundin the cytoplasm or nucleus of a biological cell, and may includecomplexes of one or more intracellular proteins and a surface membranereceptor. Exemplary intracellular proteins that may be part of suchcomplexes include, but are not limit to, PI3K proteins, Grb2 proteins,Grb7 proteins, Shc proteins, and Sos proteins, Src proteins, Cblproteins, PLCγ proteins, Shp2 proteins, GAP proteins, Nck proteins, Vavproteins, and Crk proteins. In one aspect, such complexes include PI3Kor Shc proteins. In another aspect, a complex is a stable aggregatecomprising two proteins, or from 2 to 4 proteins, or from 2 to 6proteins. As used herein, a “signaling complex” is an intracellularprotein-protein complex that is a component of a signaling pathway.

“Dimer” in reference to cell surface membrane receptors means a complexof two or more membrane-bound receptor proteins that may be the same ordifferent. Dimers of identical receptors are referred to as “homodimers”and dimers of different receptors are referred to as “heterodimers.”Dimers usually consist of two receptors in contact with one another.Dimers may be created in a cell surface membrane by passive processes,such as Van der Waal interactions, and the like, as described above inthe definition of “complex,” or dimers may be created by activeprocesses, such as by ligand-induced dimerization, covalent linkages,interaction with intracellular components, or the like, e.g.Schlessinger, Cell, 103: 211-225 (2000). As used herein, the term“dimer” is understood to refer to “cell surface membrane receptordimer,” unless understood otherwise from the context.

“Isolated” in reference to a polypeptide or protein means substantiallyseparated from the components of its natural environment. Preferably, anisolated polypeptide or protein is a composition that consists of atleast eighty percent of the polypeptide or protein identified bysequence on a weight basis as compared to components of its naturalenvironment; more preferably, such composition consists of at leastninety-five percent of the polypeptide or protein identified by sequenceon a weight basis as compared to components of its natural environment;and still more preferably, such composition consists of at leastninety-nine percent of the polypeptide or protein identified by sequenceon a weight basis as compared to components of its natural environment.Most preferably, an isolated polypeptide or protein is a homogeneouscomposition that can be resolved as a single spot after conventionalseparation by two-dimensional gel electrophoresis based on molecularweight and isoelectric point. Protocols for such analysis byconventional two-dimensional gel electrophoresis are well known to oneof ordinary skill in the art, e.g. Hames and Rickwood, Editors, GelElectrophoresis of Proteins: A Practical Approach (IRL Press, Oxford,1981); Scopes, Protein Purification (Springer-Verlag, New York, 1982);Rabilloud, Editor, Proteome Research: Two-Dimensional GelElectrophoresis and Identification Methods (Springer-Verlag, Berlin,2000).

“Kit” refers to any delivery system for delivering materials or reagentsfor carrying out a method of the invention. In the context of reactionassays, such delivery systems include systems that allow for thestorage, transport, or delivery of reaction reagents (e.g., probes,enzymes, etc. in the appropriate containers) and/or supporting materials(e.g., buffers, written instructions for performing the assay etc.) fromone location to another. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. Such contents may be delivered to theintended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains probes.

The term “ligand” is also used herein to refer to a secreted protein orprotein thereof which binds to a given receptor, through aligand-receptor interaction.

“Membrane-associated analyte” means a substance, compound, molecule, orcomponent or part of any of the foregoing that is directly or indirectlyattached to a membrane, especially a biological membrane such as thecell surface membrane of a mammalian cell or tissue. The attachment maybe direct, for example, when a membrane-associated analyte has alipophilic moiety, or is attached to another molecule that has alipophilic moiety, capable of anchoring it in a membrane. The attachmentmay also be indirect, for example, when a membrane-associated analyte isa soluble ligand that binds to, and forms a stable complex with, a cellsurface receptor. A membrane-associated analyte may be, but is notlimited to, a peptide, protein, polynucleotide, polypeptide,oligonucleotide, organic molecule, hapten, epitope, part of a biologicalcell, a posttranslational modification of a protein, a receptor, acomplex sugar attached to a membrane component such as a receptor, asoluble compound forming a stable complex with a membrane such as avitamin, a hormone, a cytokine, or the like, forming and the like. Theremay be more than one analyte associated with a single molecular entity,e.g. different phosphorylation sites on the same protein.Membrane-associated analytes include cell surface molecules, such ascell membrane receptors. In one aspect of the invention,membrane-associated analytes are cell membrane receptors selected fromthe group consisting of epidermal growth factor receptors and G-proteincoupled receptors. In particular, epidermal growth factor receptorsinclude Her1, Her2, Her3, and Her4 receptors, e.g. Yarden (cited above);Yarden and Sliwkowski (cited above). “Dimer” in reference tomembrane-associated analytes means a stable, usually non-covalent,association of two membrane-associated analytes. A dimer ofmembrane-associated analytes may form as the result of interaction witha ligand, i.e. ligand-induced dimerization, e.g. Schlessinger, Cell,110: 669-672 (2002). “Oligomer” in reference to membrane-associatedanalytes means a stable, usually non-covalent, association of at leasttwo membrane-associated analytes.

“Polypeptide” refers to a class of compounds composed of amino acidresidues chemically bonded together by amide linkages with eliminationof water between the carboxy group of one amino acid and the amino groupof another amino acid. A polypeptide is a polymer of amino acidresidues, which may contain a large number of such residues. Peptidesare similar to polypeptides, except that, generally, they are comprisedof a lesser number of amino acids. Peptides are sometimes referred to asoligopeptides. There is no clear-cut distinction between polypeptidesand peptides. For convenience, in this disclosure and claims, the term“polypeptide” will be used to refer generally to peptides andpolypeptides. The amino acid residues may be natural or synthetic.

“Protein” refers to a polypeptide, usually synthesized by a biologicalcell, folded into a defined three-dimensional structure. Proteins aregenerally from about 5,000 to about 5,000,000 or more in molecularweight, more usually from about 5,000 to about 1,000,000 molecularweight, and may include post-translational modifications, suchacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, farnesylation,demethylation, formation of covalent cross-links, formation of cystine,formation of pyroglutamate, formylation, gamma-carboxylation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, phosphorylation, prenylation,racemization, selenoylation, sulfation, and ubiquitination, e.g. Wold,F., Post-translational Protein Modifications: Perspectives andProspects, pgs. 1-12 in Post-translational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, 1983. In oneaspect, post-translational modifications are usually phosphylations ofproteins that are components of a signaling pathway. Proteins include,by way of illustration and not limitation, cytokines or interleukins,enzymes such as, e.g., kinases, proteases, galactosidases and so forth,protamines, histones, albumins, immunoglobulins, scleroproteins,phosphoproteins, mucoproteins, chromoproteins, lipoproteins,nucleoproteins, glycoproteins, T-cell receptors, proteoglycans, and thelike.

“Receptor complex” means a complex that comprises at least one cellsurface membrane receptor. Receptor complexes may include a dimer ofcell surface membrane receptors, or one or more intracellular proteins,such as adaptor proteins, that form links in the various signalingpathways. Exemplary intracellular proteins that may be part of areceptor complex includes, but is not limit to, PI3K proteins, Grb2proteins, Grb7 proteins, Shc proteins, and Sos proteins, Src proteins,Cbl proteins, PLCγ proteins, Shp2 proteins, GAP proteins, Nck proteins,Vav proteins, and Crk proteins. In one aspect, receptor complexesinclude PI3K or Shc proteins.

“Receptor tyrosine kinase,” or “RTK,” means a human receptor proteinhaving intracellular kinase activity and being selected from the RTKfamily of proteins described in Schlessinger, Cell, 103: 211-225 (2000);and Blume-Jensen and Hunter (cited above). “Receptor tyrosine kinasedimer” means a complex in a cell surface membrane comprising tworeceptor tyrosine kinase proteins. In some aspects, a receptor tyrosinekinase dimer may comprise two covalently linked receptor tyrosine kinaseproteins. Exemplary RTK dimers are listed in Table I. RTK dimers ofparticular interest are Her receptor dimers and VEGFR dimers.

“Sample” or “tissue sample” or “patient sample” or “patient cell ortissue sample” or “specimen” each means a collection of similar cellsobtained from a tissue of a subject or patient. The source of the tissuesample may be solid tissue as from a fresh, frozen and/or preservedorgan or tissue sample or biopsy or aspirate; blood or any bloodconstituents; bodily fluids such as cerebral spinal fluid, amnioticfluid, peritoneal fluid, or interstitial fluid; or cells from any timein gestation or development of the subject. The tissue sample maycontain compounds which are not naturally intermixed with the tissue innature such as preservatives, anticoagulants, buffers, fixatives,nutrients, antibiotics, or the like. In one aspect of the invention,tissue samples or patient samples are fixed, particularly conventionalformalin-fixed paraffin-embedded samples. Such samples are typicallyused in an assay for receptor complexes in the form of thin sections,e.g. 3-10 μm thick, of fixed tissue mounted on a microscope slide, orequivalent surface. Such samples also typically undergo a conventionalre-hydration procedure, and optionally, an antigen retrieval procedureas a part of, or preliminary to, assay measurements.

“Separation profile” in reference to the separation of molecular tagsmeans a chart, graph, curve, bar graph, or other representation ofsignal intensity data versus a parameter related to the molecular tags,such as retention time, mass, or the like, that provides a readout, ormeasure, of the number of molecular tags of each type produced in anassay. A separation profile may be an electropherogram, a chromatogram,an electrochromatogram, a mass spectrogram, or like graphicalrepresentation of data depending on the separation technique employed. A“peak” or a “band” or a “zone” in reference to a separation profilemeans a region where a separated compound is concentrated. There may bemultiple separation profiles for a single assay if, for example,different molecular tags have different fluorescent labels havingdistinct emission spectra and data is collected and recorded at multiplewavelengths. In one aspect, released molecular tags are separated bydifferences in electrophoretic mobility to form an electropherogramwherein different molecular tags correspond to distinct peaks on theelectropherogram. A measure of the distinctness, or lack of overlap, ofadjacent peaks in an electropherogram is “electrophoretic resolution,”which may be taken as the distance between adjacent peak maximumsdivided by four times the larger of the two standard deviations of thepeaks. Preferably, adjacent peaks have a resolution of at least 1.0, andmore preferably, at least 1.5, and most preferably, at least 2.0. In agiven separation and detection system, the desired resolution may beobtained by selecting a plurality of molecular tags whose members haveelectrophoretic mobilities that differ by at least a peak-resolvingamount, such quantity depending on several factors well known to thoseof ordinary skill, including signal detection system, nature of thefluorescent moieties, the diffusion coefficients of the tags, thepresence or absence of sieving matrices, nature of the electrophoreticapparatus, e.g. presence or absence of channels, length of separationchannels, and the like. Electropherograms may be analyzed to associatefeatures in the data with the presence, absence, or quantities ofmolecular tags using analysis programs, such as disclosed in Williams etal, U.S. patent publication 2003/0170734 A1.

“Specific” or “specificity” in reference to the binding of one moleculeto another molecule, such as a binding compound, or probe, for a targetanalyte or complex, means the recognition, contact, and formation of astable complex between the probe and target, together with substantiallyless recognition, contact, or complex formation of the probe with othermolecules. In one aspect, “specific” in reference to the binding of afirst molecule to a second molecule means that to the extent the firstmolecule recognizes and forms a complex with another molecules in areaction or sample, it forms the largest number of the complexes withthe second molecule. In one aspect, this largest number is at leastfifty percent of all such complexes form by the first molecule.Generally, molecules involved in a specific binding event have areas ontheir surfaces or in cavities giving rise to specific recognitionbetween the molecules binding to each other. Examples of specificbinding include antibody-antigen interactions, enzyme-substrateinteractions, formation of duplexes or triplexes among polynucleotidesand/or oligonucleotides, receptor-ligand interactions, and the like.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and kits for detecting and/ormeasuring dimers and/or oligomers of cell surface receptors,particularly homodimers or homo-oligomers of cell surface receptors.

In one aspect of the invention, homodimeric complexes may be measured asillustrated in FIG. 1A. An assay may comprise three reagents (1128):cleaving probes (1134), first binding compound (1130), and secondbinding compound (1132). First binding compound (1130) and cleavingprobe (1134) are constructed to be specific for the same antigenicdeterminant (1135) on protein (1138) that exists in a membrane as eithera homodimer (1136) or a monomer (1138). After reagents (1128) arecombined with a sample under conditions that promote the formation ofstable complexes between the reagents and their respective targets,multiple complexes (1142 through 1150) form in the assay mixture.Because cleaving probe (1134) and binding compound (1130) are specificfor the same antigenic determinant (1135), four different combinations(1144 through 1150) of reagents may form complexes with homodimers. Ofthe complexes in the assay mixture, only those (1143) with both acleaving probe (1134) and at least one binding compound will contributereleased molecular tags (1151) for separation and detection (1154). Inthis embodiment, the size of peak (1153) is proportional to the amountof homodimer in the assay mixture, while the size of peak (1152) isproportional to the total amount of protein (1138) in the assay mixture,both in monomeric form (1142) or in homodimeric form (1146 and 1148).FIG. 1B illustrates the analogous measurements for cell surfacereceptors that form heterodimers in a cell surface membrane. One skilledin the art would understand that dimmers may be measured in eitherlysates of cells or tissues, or in fixed samples whose membranes havebeen permeabilized or removed by the fixing process. In such cases,binding compounds may be specific for either extracellular orintracellular domains of cell surface membrane receptors. Optionally,prior to illumination the binding buffer may be removed and replacedwith a buffer more suitable for separation, i.e. a separation buffer.For example, binding buffers typically have salt concentrations that maydegrade the performance of some separation techniques, such as capillaryelectrophoresis, for separating molecular tags into distinct peaks. Inone embodiment, such exchange of buffers may be accomplished by membranefiltration.

As mentioned above, a method of measuring signaling complexes comprisingheterodimers is illustrated in FIG. 1B. Heterodimeric complex (100)forms by the binding of proteins (104) and (102), e.g. Akt and PDK 1.Reagents (122) of the invention, comprising cleaving probes (108) (inthis illustration having photosensitizer “PS” attached) and bindingcompounds (106), are mixed (109) with a sample containing complex (100)under conditions that permit the specific binding (112) of cleavingprobes (108) and binding compounds (106) to their respective antigenicdeterminants on complex (100) that are on different proteins of thecomplex. After binding, and optionally washing or buffer exchange,cleaving probes (108) are activated to generate an active species that,e.g. in the case of singlet oxygen, diffuses out from a photosensitizersto an effective proximity (110). Cleavable linkages within thisproximity are cleaved and molecular tags are released (114). Releasedmolecular tags (116) are then separated (117) and a separation profile(120), such as an electropherogram, is produced, in which peak (118) isidentified and correlated to molecular tag, “mT₁” and peak (124) isidentified and correlated with molecular tag, “mT₂.” By employingadditional binding compounds and molecular tags, additional complexesmay be measured. As with the ratiometric measure of an activatedeffector protein, the amount of heterodimeric complexes may be providedas a ratio of peak areas. FIG. 1D illustrates the analogous measurementsfor cell surface receptors that form heterodimers in cell surfacemembrane (161).

In some embodiments of the invention, ratiometric measurements may bemade on effector proteins or receptor components havingpost-translational modifications, such as phosphoylation, as illustratedin FIG. 1C. Effector protein or receptor component (10) exists in twostates in a cell, one having a post-translational modification, e.g.such as a phosphate group (12), and the other not having such apost-translational modification. Reagents (14) of the invention,comprising cleaving probes (18) (in this illustration havingphotosensitizer “PS” attached) and binding compounds (16), are mixed(19) with a sample containing both the activated and inactivated formsof effector protein or receptor component (10) under conditions thatpermit the specific binding of cleaving probes (18) and bindingcompounds (16) to their respective antigenic determinants on theactivated and inactivated forms of effector protein or receptorcomponent (10) resulting in the formation of either complex (21) orcomplex (23). After binding, and optionally washing or buffer exchange,cleaving probes (18) are activated to generate an active species that,e.g. in the case of singlet oxygen, diffuses out from a photosensitizersto an effective proximity (20). Cleavable linkages within this proximityare cleaved and molecular tags are released (22). Released moleculartags (22) are then separated (25) and a separation profile (28), such asan electropherogram, is produced, in which peak (24) is identified andcorrelated to molecular tag, “mT₁” and peak (26) is identified andcorrelated to molecular tag, “mT₂.” In one aspect, a ratiometric measureof activated effector protein or receptor component (10) is provided asthe ratio of areas of peaks (24) and (26).

Another aspect of the invention is illustrated in FIGS. 1E and 1F, whichprovides for the simultaneous detection or measurement of multiplecomplexes, dimers, and activated effector proteins in a cellular sample.Cells (160), which may be from a sample from in vitro cultures or from aspecimen of patient tissue, are lysed (172) to form lysate (174) inwhich cellular components are rendered accessible, such componentsincluding molecular complexes associated with the cell membrane (173),and/or within the cytosol (179), and/or within the cell nucleus.Complexes associated with signaling pathways include, but are notlimited to, surface receptor complexes, such as receptor dimers (162 or170), receptor complexes including adaptor or scaffold molecules ofvarious types (162, 168, or 170), dimers and higher order complexes ofintracellular proteins (164), phosphorylation sites of proteins in suchcomplexes (166), phosphorylated effector proteins (163), and the like.After lysing, the resulting lysate (174) is combined with assay reagents(176) that include multiple cleaving probes (175) and multiple bindingcompounds (177). Assay conditions are selected (178) that allow reagents(176) to specifically bind to their respective targets, so that uponactivation cleavable linkages within the effective proximity (180) ofthe cleavage-inducing moieties are cleaved and molecular tags arereleased (182). Also illustrated are intracellular complexes, e.g.signaling complexes (181), receptor dimers (183), and effector proteins(185). As above, after cleavage, the released molecular tags areseparated (184) and identified in a separation profile (186), such as anelectropherogram, and based on the number and quantities of moleculartags measured, a profile is obtained of the selected molecularcomplexes, RTK dimers, and/or effector proteins in the cells of thesample. One skilled in the art would understand that dimers may bemeasured in either lysates of cells or tissues, or in fixed sampleswhose membranes have been permeabilized or removed by the fixingprocess. In such cases, binding compounds may be specific for eitherextracellular or intracellular domains of cell surface membranereceptors.

FIGS. 1G and 1H illustrate an embodiment of the invention for measuringreceptor complexes in fixed or frozen tissue samples. Fixed tissuesample (1000), e.g. a formalin-fixed paraffin-embedded sample, is slicedto provide a section (1004) using a microtome, or like instrument, whichafter placing on surface (1006), which may be a microscope slide, it isde-waxed and re-hydrated for application of assay reagents. Enlargement(1007) shows portion (1008) of section (1004) on portion (1014) ofmicroscope slide (1006). Receptor dimer molecules (1018) are illustratedas embedded in the remnants of membrane structure (1016) of the fixedsample. In accordance with this aspect of the invention, cleaving probeand binding compounds are incubated with the fixed sample so that theybind to their target molecules. For example, cleaving probes(1012)(illustrated in the figure as an antibody having a photosensitizer(“PS”) attached) and first binding compound (1010)(illustrated as anantibody having molecular tag “mT₁₁” attached) specifically bind toreceptor (1011) common to all of the dimers shown, second bindingcompound (1017)(with “mT₂”) specifically binds to receptor (1015), andthird binding compound (1019)(with “mT₃”) specifically binds to receptor(1013). After washing to remove binding compounds and cleaving probethat are not specifically bound to their respective target molecules,buffer (1024) (referred to as “illumination buffer” in the figure) isadded. For convenience, buffer (1024) may be contained on section(1004), or a portion thereof, by creating a hydrophobic barrier on slide(1006), e.g. with a wax pen. After illumination of photosensitizers andrelease of molecular tags (1026), buffer (1024) now containing releasemolecular tags (1025) is transferred to a separation device, such as acapillary electrophoresis instrument, for separation (1028) andidentification of the released molecular tags in, for example,electropherogram (1030). Although the illustrations of FIGS. 1G and 1Hdescribe measurement of heterodimers, one of ordinary skill in the artwould appreciate that homodimers can be measured using similartechniques following the process described above, e.g. in relation toFIG. 1A.

Measurements made directly on tissue samples, particularly asillustrated in FIGS. 1G and 1H, may be normalized by includingmeasurements on cellular or tissue targets that are representative ofthe total cell number in the sample and/or the numbers of particularsubtypes of cells in the sample. The additional measurement may bepreferred, or even necessary, because of the cellular and tissueheterogeneity in patient samples, particularly tumor samples, which maycomprise substantial fractions of normal cells. For example, in FIG. 1H,values for the total amount of receptor (1011) may be given as a ratioof the following two measurements: area of peak (1032) of molecular tag(“mT₁”) and the area of a peak corresponding to a molecular tagcorrelated with a cellular or tissue component common to all the cellsin the sample, e.g. tubulin, or the like. In some cases, where all thecells in the sample are epithelial cells, cytokeratin may be used.Accordingly, detection methods based on releasable molecular tags mayinclude an additional step of providing a binding compound (with adistinct molecular tag) specific for a normalization protein, such astubulin.

Preparation of Samples

Samples containing molecular complexes may come from a wide variety ofsources for use with the present invention to relate receptor complexespopulations to disease status or health status, including cell cultures,animal or plant tissues, patient biopsies, or the like. Preferably,samples are human patient samples. Samples are prepared for assays ofthe invention using conventional techniques, which may depend on thesource from which a sample is taken. A. Solid Tissue Samples. Forbiopsies and medical specimens, guidance is provided in the followingreferences: Bancroft J D & Stevens A, eds. Theory and Practice ofHistological Techniques (Churchill Livingstone, Edinburgh, 1977);Pearse, Histochemistry. Theory and applied. 4^(th) ed. (ChurchillLivingstone, Edinburgh, 1980).

In the area of cancerous disease status, examples of patient tissuesamples that may be used include, but are not limited to, breast,prostate, ovary, colon, lung, endometrium, stomach, salivary gland orpancreas. The tissue sample can be obtained by a variety of proceduresincluding, but not limited to surgical excision, aspiration or biopsy.The tissue may be fresh or frozen. In one embodiment, assays of theinvention are carried out on tissue samples that have been fixed andembedded in paraffin or the like; therefore, in such embodiments a stepof deparaffination is carried out. A tissue sample may be fixed (i.e.preserved) by conventional methodology [See e.g., “Manual ofHistological Staining Method of the Armed Forces Institute ofPathology,” 3^(rd) edition (1960) Lee G. Luna, HT (ASCP) Editor, TheBlakston Division McGraw-Hill Book Company, New York; The Armed ForcesInstitute of Pathology Advanced Laboratory Methods in Histology andPathology (1994) Ulreka V. Mikel, Editor, Armed Forces Institute ofPathology, American Registry of Pathology, Washington, D.C. One of skillin the art will appreciate that the choice of a fixative is determinedby the purpose for which the tissue is to be histologically stained orotherwise analyzed. One of skill in the art will also appreciate thatthe length of fixation depends upon the size of the tissue sample andthe fixative used. By way of example, neutral buffered formalin, Bouin'sor paraformaldehyde, may be used to fix a tissue sample.

Generally, a tissue sample is first fixed and is then dehydrated throughan ascending series of alcohols, infiltrated and embedded with paraffinor other sectioning media so that the tissue sample may be sectioned.Alternatively, one may section the tissue and fix the sections obtained.By way of example, the tissue sample may be embedded and processed inparaffin by conventional methodology (See e.g., “Manual of HistologicalStaining Method of the Armed Forces Institute of Pathology”, supra).Examples of paraffin that may be used include, but are not limited to,Paraplast, Broloid, and Tissuemay. Once the tissue sample is embedded,the sample may be sectioned by a microtome or the like (See e.g.,“Manual of Histological Staining Method of the Armed Forces Institute ofPathology”, supra). By way of example for this procedure, sections mayhave a thickness in a range from about three microns to about twelvemicrons, and preferably, a thickness in a range of from about 5 micronsto about 10 microns. In one aspect, a section may have an area of fromabout 10 mm² to about 1 cm². Once cut, the sections may be attached toslides by several standard methods. Examples of slide adhesives include,but are not limited to, silane, gelatin, poly-L-lysine and the like. Byway of example, the paraffin embedded sections may be attached topositively charged slides and/or slides coated with poly-L-lysine.

If paraffin has been used as the embedding material, the tissue sectionsare generally deparaffinized and rehydrated to water. The tissuesections may be deparaffinized by several conventional standardmethodologies. For example, xylenes and a gradually descending series ofalcohols may be used (See e.g., “Manual of Histological Staining Methodof the Armed Forces Institute of Pathology”, supra). Alternatively,commercially available deparaffinizing non-organic agents such asHemo-De® (CMS, Houston, Tex.) may be used.

For mammalian tissue culture cells, fresh tissues, or like sources,samples may be prepared by conventional cell lysis techniques (e.g. 0.14M NaCl, 1.5 mM MgCl₂, 10 mM Tris-Cl (pH 8.6), 0.5% Nonidet P-40, andprotease and/or phosphatase inhibitors as required). For fresh mammaliantissues, sample preparation may also include a tissue disaggregationstep, e.g. crushing, mincing, grinding, sonication, or the like.

B. Magnetic Isolation of Cells. In some applications, such as measuringdimers on rare metastatic cells from a patient's blood, an enrichmentstep may be carried out prior to conducting an assay for surfacereceptor dimer populations. Immunomagnetic isolation or enrichment maybe carried out using a variety of techniques and materials known in theart, as disclosed in the following representative references that areincorporated by reference: Terstappen et al, U.S. Pat. No. 6,365,362;Terstappen et al, U.S. Pat. No. 5,646,001; Rohr et al, U.S. Pat. No.5,998,224; Kausch et al, U.S. Pat. No. 5,665,582; Kresse et al, U.S.Pat. No. 6,048,515; Kausch et al, U.S. Pat. No. 5,508,164; Miltenyi etal, U.S. Pat. No. 5,691,208; Molday, U.S. Pat. No. 4,452,773; Kronick,U.S. Pat. No. 4,375,407; Radbruch et al, chapter 23, in Methods in CellBiology, Vol, 42 (Academic Press, New York, 1994); Uhlen et al, Advancesin Biomagnetic Separation (Eaton Publishing, Natick, 1994); Safarik etal, J. Chromatography B, 722: 33-53 (1999); Miltenyi et al, Cytometry,11: 231-238 (1990); Nakamura et al, Biotechnol. Prog., 17: 1145-1155(2001); Moreno et al, Urology, 58: 386-392 (2001); Racila et al, Proc.Natl. Acad. Sci., 95: 4589-4594 (1998); Zigeuner et al, J. Urology, 169:701-705 (2003); Ghossein et al, Seminars in Surgical Oncology, 20:304-311 (2001).

The preferred magnetic particles for use in carrying out this inventionare particles that behave as colloids. Such particles are characterizedby their sub-micron particle size, which is generally less than about200 nanometers (nm) (0.20 microns), and their stability to gravitationalseparation from solution for extended periods of time. In addition tothe many other advantages, this size range makes them essentiallyinvisible to analytical techniques commonly applied to cell analysis.Particles within the range of 90-150 nm and having between 70-90%magnetic mass are contemplated for use in the present invention.Suitable magnetic particles are composed of a crystalline core ofsuperparamagnetic material surrounded by molecules which are bonded,e.g., physically absorbed or covalently attached, to the magnetic coreand which confer stabilizing colloidal properties. The coating materialshould preferably be applied in an amount effective to prevent nonspecific interactions between biological macromolecules found in thesample and the magnetic cores. Such biological macromolecules mayinclude sialic acid residues on the surface of non-target cells,lectins, glyproteins and other membrane components. In addition, thematerial should contain as much magnetic mass/nanoparticle as possible.The size of the magnetic crystals comprising the core is sufficientlysmall that they do not contain a complete magnetic domain. The size ofthe nanoparticles is sufficiently small such that their Brownian energyexceeds their magnetic moment. As a consequence, North Pole, South Polealignment and subsequent mutual attraction/repulsion of these colloidalmagnetic particles does not appear to occur even in moderately strongmagnetic fields, contributing to their solution stability. Finally, themagnetic particles should be separable in high magnetic gradientexternal field separators. That characteristic facilitates samplehandling and provides economic advantages over the more complicatedinternal gradient columns loaded with ferromagnetic beads or steel wool.Magnetic particles having the above-described properties can be preparedby modification of base materials described in U.S. Pat. Nos. 4,795,698,5,597,531 and 5,698,271, which patents are incorporated by reference.

Assays Using Releasable Molecular Tags

Many advantages are provided by measuring dimer populations usingreleasable molecular tags, including (1) separation of releasedmolecular tags from an assay mixture provides greatly reduced backgroundand a significant gain in sensitivity; and (2) the use of molecular tagsthat are specially designed for ease of separation and detectionprovides a convenient multiplexing capability so that multiple receptorcomplex components may be readily measured simultaneously in the sameassay. Assays employing such tags can have a variety of forms and aredisclosed in the following references: Singh et al, U.S. Pat. No.6,627,400; U.S. patent publications Singh et al, 2002/0013126; and2003/0170915, and Williams et al, 2002/0146726; and Chan-Hui et al,International patent publication WO 2004/011900, all of which areincorporated herein by reference. For example, a wide variety ofseparation techniques may be employed that can distinguish moleculesbased on one or more physical, chemical, or optical differences amongmolecules being separated including but not limited to electrophoreticmobility, molecular weight, shape, solubility, pKa, hydrophobicity,charge, charge/mass ratio, polarity, or the like. In one aspect,molecular tags in a plurality or set differ in electrophoretic mobilityand optical detection characteristics and are separated byelectrophoresis. In another aspect, molecular tags in a plurality or setmay differ in molecular weight, shape, solubility, pKa, hydrophobicity,charge, polarity, and are separated by normal phase or reverse phaseHPLC, ion exchange HPLC, capillary electrochromatography, massspectroscopy, gas phase chromatography, or like technique.

Sets of molecular tags are provided that are separated into distinctbands or peaks by a separation technique after they are released frombinding compounds. Identification and quantification of such peaksprovides a measure or profile of the kinds and amounts of receptordimers. Molecular tags within a set may be chemically diverse; however,for convenience, sets of molecular tags are usually chemically related.For example, they may all be peptides, or they may consist of differentcombinations of the same basic building blocks or monomers, or they maybe synthesized using the same basic scaffold with different substituentgroups for imparting different separation characteristics, as describedmore fully below. The number of molecular tags in a plurality may varydepending on several factors including the mode of separation employed,the labels used on the molecular tags for detection, the sensitivity ofthe binding moieties, the efficiency with which the cleavable linkagesare cleaved, and the like. In one aspect, the number of molecular tagsin a plurality for measuring populations of receptor dimers is in therange of from 2 to 10. In other aspects, the size of the plurality maybe in the range of from 2 to 8, 2 to 6, 2 to 4, or 2 to 3.

Receptor dimers may be detected in assays having homogeneous formats ora non-homogeneous, i.e. heterogeneous, formats. In a homogeneous format,no step is required to separate binding compounds specifically bound totarget complexes from unbound binding compounds. In a preferredembodiment, homogeneous formats employ reagent pairs comprising (i) oneor more binding compounds with releasable molecular tags and (ii) atleast one cleaving probe that is capable of generating an active speciesthat reacts with and releases molecular tags within an effectiveproximity of the cleaving probe.

Receptor dimers may also be detected by assays employing a heterogeneousformat. Heterogeneous techniques normally involve a separation step,where intracellular complexes having binding compounds specificallybound are separated from unbound binding compounds, and optionally,other sample components, such as proteins, membrane fragments, and thelike. Separation can be achieved in a variety of ways, such as employinga reagent bound to a solid support that distinguishes betweencomplex-bound and unbound binding compounds. The solid support may be avessel wall, e.g., microtiter well plate well, capillary, plate, slide,beads, including magnetic beads, liposomes, or the like. The primarycharacteristics of the solid support are that it (1) permits segregationof the bound and unbound binding compounds and (2) does not interferewith the formation of the binding complex, or the other operations inthe determination of receptor dimers. Usually, in fixed samples, unboundbinding compounds are removed simply by washing.

With detection using molecular tags in a heterogeneous format, afterwashing, a sample may be combined with a solvent into which themolecular tags are to be released. Depending on the nature of thecleavable bond and the method of cleavage, the solvent may include anyadditional reagents for the cleavage. Where reagents for cleavage arenot required, the solvent conveniently may be a separation buffer, e.g.an electrophoretic separation medium. For example, where the cleavablelinkage is photolabile or cleavable via an active species generated by aphotosensitizer, the medium may be irradiated with light of appropriatewavelength to release the molecular tags into the buffer.

In either format, if the assay reaction conditions interfere with theseparation technique employed, it may be necessary to remove, orexchange, the assay reaction buffer prior to cleavage and separation ofthe molecular tags. For example, in some embodiments, assay conditionsinclude salt concentrations (e.g. required for specific binding) thatdegrade separation performance when molecular tags are separated on thebasis of electrophoretic mobility. In such embodiments, an assay bufferis replaced by a separation buffer, or medium, prior to release andseparation of the molecular tags.

Assays employing releasable molecular tags and cleaving probes can bemade in many different formats and configurations depending on thecomplexes that are detected or measured. Based on the presentdisclosure, it is a design choice for one of ordinary skill in the artto select the numbers and specificities of particular binding compoundsand cleaving probes.

A. Binding Compounds

As mentioned above, mixtures containing pluralities of different bindingcompounds may be provided, wherein each different binding compound hasone or more molecular tags attached through cleavable linkages. Thenature of the binding compound, cleavable linkage and molecular tag mayvary widely. A binding compound may comprise an antibody bindingcomposition, an antibody, a peptide, a peptide or non-peptide ligand fora cell surface receptor, a protein, an oligonucleotide, anoligonucleotide analog, such as a peptide nucleic acid, a lectin, or anyother molecular entity that is capable of specific binding or stablecomplex formation with an analyte of interest, such as a complex ofproteins. In one aspect, a binding compound, which can be represented bythe formula below, comprises one or more molecular tags attached to abinding moiety.

B-(L-E)_(k)

wherein B is binding moiety; L is a cleavable linkage; and E is amolecular tag. In homogeneous assays, cleavable linkage, L, may be anoxidation-labile linkage, and more preferably, it is a linkage that maybe cleaved by singlet oxygen. The moiety “-(L-E)_(k)” indicates that asingle binding compound may have multiple molecular tags attached viacleavable linkages. In one aspect, k is an integer greater than or equalto one, but in other embodiments, k may be greater than several hundred,e.g. 100 to 500, or k is greater than several hundred to as many asseveral thousand, e.g. 500 to 5000. Usually each of the plurality ofdifferent types of binding compound has a different molecular tag, E.Cleavable linkages, e.g. oxidation-labile linkages, and molecular tags,E, are attached to B by way of conventional chemistries.

Preferably, B is an antibody binding composition that specifically bindsto a target, such as a predetermined antigenic determinant of a targetprotein, such as a cell surface receptor. Such compositions are readilyformed from a wide variety of commercially available antibodies, bothmonoclonal and polyclonal, specific for proteins of interest. Inparticular, antibodies specific for epidermal growth factor receptorsare disclosed in the following patents, which are incorporated byreferences: U.S. Pat. Nos. 5,677,171; 5,772,997; 5,968,511; 5,480,968;5,811,098. U.S. Pat. No. 6,488,390, incorporated herein by reference,discloses antibodies specific for a G-protein coupled receptor, CCR4.U.S. Pat. No. 5,599,681, incorporated herein by reference, disclosesantibodies specific for phosphorylation sites of proteins. Commercialvendors, such as Cell Signaling Technology (Beverly, Mass.), BiosourceInternational (Camarillo, Calif.), and Upstate (Charlottesville, Va.),also provide monoclonal and polyclonal antibodies specific for manyreceptors.

Cleavable linkage, L, can be virtually any chemical linking group thatmay be cleaved under conditions that do not degrade the structure oraffect detection characteristics of the released molecular tag, E.Whenever a cleaving probe is used in a homogeneous assay format,cleavable linkage, L, is cleaved by a cleavage agent generated by thecleaving probe that acts over a short distance so that only cleavablelinkages in the immediate proximity of the cleaving probe are cleaved.Typically, such an agent must be activated by making a physical orchemical change to the reaction mixture so that the agent produces ashort lived active species that diffuses to a cleavable linkage toeffect cleavage. In a homogeneous format, the cleavage agent ispreferably attached to a binding moiety, such as an antibody, thattargets prior to activation the cleavage agent to a particular site inthe proximity of a binding compound with releasable molecular tags. Insuch embodiments, a cleavage agent is referred to herein as a“cleavage-inducing moiety,” which is discussed more fully below.

In a non-homogeneous format, because specifically bound bindingcompounds are separated from unbound binding compounds, a widerselection of cleavable linkages and cleavage agents are available foruse. Cleavable linkages may not only include linkages that are labile toreaction with a locally acting reactive species, such as hydrogenperoxide, singlet oxygen, or the like, but also linkages that are labileto agents that operate throughout a reaction mixture, such asbase-labile linkages, photocleavable linkages, linkages cleavable byreduction, linkages cleaved by oxidation, acid-labile linkages, peptidelinkages cleavable by specific proteases, and the like. Referencesdescribing many such linkages include Greene and Wuts, Protective Groupsin Organic Synthesis, Second Edition (John Wiley & Sons, New York,1991); Hermanson, Bioconjugate Techniques (Academic Press, New York,1996); and Still et al, U.S. Pat. No. 5,565,324.

In one aspect, commercially available cleavable reagent systems may beemployed with the invention. For example, a disulfide linkage may beintroduced between an antibody binding composition and a molecular tagusing a heterofunctional agent such as N-succinimidyl3-(2-pyridyldithio)propionate (SPDP),succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT), orthe like, available from vendors such as Pierce Chemical Company(Rockford, Ill.). Disulfide bonds introduced by such linkages can bebroken by treatment with a reducing agent, such as dithiothreitol (DTT),dithioerythritol (DTE), 2-mercaptoethanol, sodium borohydride, or thelike. Typical concentrations of reducing agents to effect cleavage ofdisulfide bonds are in the range of from 10 to 100 mM. An oxidativelylabile linkage may be introduced between an antibody binding compositionand a molecular tag using the homobifunctional NHS ester cross-linkingreagent, disuccinimidyl tartarate (DST)(available from Pierce) thatcontains central cis-diols that are susceptible to cleavage with sodiumperiodate (e.g., 15 mM periodate at physiological pH for 4 hours).Linkages that contain esterified spacer components may be cleaved withstrong nucleophilic agents, such as hydroxylamine, e.g. 0.1 Nhydroxylamine, pH 8.5, for 3-6 hours at 37° C. Such spacers can beintroduced by a homobifunctional cross-linking agent such as ethyleneglycol bis(succinimidylsuccinate)(EGS) available from Pierce (Rockford,Ill.). A base labile linkage can be introduced with a sulfone group.Homobifunctional cross-linking agents that can be used to introducesulfone groups in a cleavable linkage includebis[2-(succinimidyloxycarbonyloxy) ethyl]sulfone (BSOCOES), and4,4-difluoro-3,3-dinitrophenylsulfone (DFDNPS). Exemplary basicconditions for cleavage include 0.1 M sodium phosphate, adjusted to pH11.6 by addition of Tris base, containing 6 M urea, 0.1% SDS, and 2 mMDTT, with incubation at 37° C. for 2 hours. Photocleavable linkagesinclude those disclosed in Rothschild et al, U.S. Pat. No. 5,986,076.

When L is oxidation labile, L may be a thioether or its selenium analog;or an olefin, which contains carbon-carbon double bonds, whereincleavage of a double bond to an oxo group, releases the molecular tag,E. Illustrative oxidation labile linkages are disclosed in Singh et al,U.S. Pat. No. 6,627,400; and U.S. patent publications Singh et al,2002/0013126; and 2003/0170915, and in Willner et al, U.S. Pat. No.5,622,929, all of which are incorporated herein by reference.

Molecular tag, E, in the present invention may comprise an electrophorictag as described in the following references when separation ofpluralities of molecular tags are carried out by gas chromatography ormass spectrometry: Zhang et al, Bioconjugate Chem., 13: 1002-1012(2002); Giese, Anal. Chem., 2: 165-168 (1983); and U.S. Pat. Nos.4,650,750; 5,360,819; 5,516,931; 5,602,273; and the like.

Molecular tag, E, is preferably a water-soluble organic compound that isstable with respect to the active species, especially singlet oxygen,and that includes a detection or reporter group. Otherwise, E may varywidely in size and structure. In one aspect, E has a molecular weight inthe range of from about 50 to about 2500 daltons, more preferably, fromabout 50 to about 1500 daltons. Preferred structures of E are describedmore fully below. E may comprise a detection group for generating anelectrochemical, fluorescent, or chromogenic signal. In embodimentsemploying detection by mass, E may not have a separate moiety fordetection purposes. Preferably, the detection group generates afluorescent signal.

Molecular tags within a plurality are selected so that each has a uniqueseparation characteristic and/or a unique optical property with respectto the other members of the same plurality. In one aspect, thechromatographic or electrophoretic separation characteristic isretention time under set of standard separation conditions conventionalin the art, e.g. voltage, column pressure, column type, mobile phase,electrophoretic separation medium, or the like. In another aspect, theoptical property is a fluorescence property, such as emission spectrum,fluorescence lifetime, fluorescence intensity at a given wavelength orband of wavelengths, or the like. Preferably, the fluorescence propertyis fluorescence intensity. For example, each molecular tag of aplurality may have the same fluorescent emission properties, but eachwill differ from one another by virtue of a unique retention time. Onthe other hand, or two or more of the molecular tags of a plurality mayhave identical migration, or retention, times, but they will have uniquefluorescent properties, e.g. spectrally resolvable emission spectra, sothat all the members of the plurality are distinguishable by thecombination of molecular separation and fluorescence measurement.

Preferably, released molecular tags are detected by electrophoreticseparation and the fluorescence of a detection group. In suchembodiments, molecular tags having substantially identical fluorescenceproperties have different electrophoretic mobilities so that distinctpeaks in an electropherogram are formed under separation conditions.Preferably, pluralities of molecular tags of the invention are separatedby conventional capillary electrophoresis apparatus, either in thepresence or absence of a conventional sieving matrix. Exemplarycapillary electrophoresis apparatus include Applied Biosystems (FosterCity, Calif.) models 310, 3100 and 3700; Beckman (Fullerton, Calif.)model P/ACE MDQ; Amersham Biosciences (Sunnyvale, Calif.) MegaBACE 1000or 4000; SpectruMedix genetic analysis system; and the like.Electrophoretic mobility is proportional to q/m^(2/3), where q is thecharge on the molecule and M is the mass of the molecule. Desirably, thedifference in mobility under the conditions of the determination betweenthe closest electrophoretic labels will be at least about 0.001, usually0.002, more usually at least about 0.01, and may be 0.02 or more.Preferably, in such conventional apparatus, the electrophoreticmobilities of molecular tags of a plurality differ by at least onepercent, and more preferably, by at least a percentage in the range offrom 1 to 10 percent. Molecular tags are identified and quantified byanalysis of a separation profile, or more specifically, anelectropherogram, and such values are correlated with the amounts andkinds of receptor dimers present in a sample. For example, during orafter electrophoretic separation, the molecular tags are detected oridentified by recording fluorescence signals and migration times (ormigration distances) of the separated compounds, or by constructing achart of relative fluorescent and order of migration of the moleculartags (e.g., as an electropherogram). Preferably, the presence, absence,and/or amounts of molecular tags are measured by using one or morestandards as disclosed by Williams et al, U.S. patent publication2003/0170734A1, which is incorporated herein by reference.

Pluralities of molecular tags may also be designed for separation bychromatography based on one or more physical characteristics thatinclude but are not limited to molecular weight, shape, solubility, pKa,hydrophobicity, charge, polarity, or the like, e.g. as disclosed in U.S.patent publication 2003/0235832, which is incorporated by reference. Achromatographic separation technique is selected based on parameterssuch as column type, solid phase, mobile phase, and the like, followedby selection of a plurality of molecular tags that may be separated toform distinct peaks or bands in a single operation. Several factorsdetermine which HPLC technique is selected for use in the invention,including the number of molecular tags to be detected (i.e. the size ofthe plurality), the estimated quantities of each molecular tag that willbe generated in the assays, the availability and ease of synthesizingmolecular tags that are candidates for a set to be used in multiplexedassays, the detection modality employed, and the availability,robustness, cost, and ease of operation of HPLC instrumentation,columns, and solvents. Generally, columns and techniques are favoredthat are suitable for analyzing limited amounts of sample and thatprovide the highest resolution separations. Guidance for making suchselections can be found in the literature, e.g. Snyder et al, PracticalHPLC Method Development, (John Wiley & Sons, New York, 1988); Millner,“High Resolution Chromatography: A Practical Approach”, OxfordUniversity Press, New York (1999), Chi-San Wu, “Column Handbook for SizeExclusion Chromatography”, Academic Press, San Diego (1999), and Oliver,“HPLC of Macromolecules: A Practical Approach, Oxford University Press”,Oxford, England (1989). In particular, procedures are available forsystematic development and optimization of chromatographic separationsgiven conditions, such as column type, solid phase, and the like, e.g.Haber et al, J. Chromatogr. Sci., 38: 386-392 (2000); Outinen et al,Eur. J. Pharm. Sci., 6: 197-205 (1998); Lewis et al, J. Chromatogr.,592: 183-195 and 197-208 (1992); and the like. An exemplary HPLCinstrumentation system suitable for use with the present invention isthe Agilent 1100 Series HPLC system (Agilent Technologies, Palo Alto,Calif.).

In one aspect, molecular tag, E, is (M, D), where M is amobility-modifying moiety and D is a detection moiety. The notation “(M,D)” is used to indicate that the ordering of the M and D moieties may besuch that either moiety can be adjacent to the cleavable linkage, L.That is, B-L-(M, D)” designates binding compound of either of two forms:“B-L-M-D” or “B-L-D-M.”

Detection moiety, D, may be a fluorescent label or dye, a chromogeniclabel or dye, an electrochemical label, or the like. Preferably, D is afluorescent dye. Exemplary fluorescent dyes for use with the inventioninclude water-soluble rhodamine dyes, fluoresceins,4,7-dichlorofluoresceins, benzoxanthene dyes, and energy transfer dyes,disclosed in the following references: Handbook of Molecular Probes andResearch Reagents, 8^(th) ed., (Molecular Probes, Eugene, 2002); Lee etal, U.S. Pat. No. 6,191,278; Lee et al, U.S. Pat. No. 6,372,907; Menchenet al, U.S. Pat. No. 6,096,723; Lee et al, U.S. Pat. No. 5,945,526; Leeet al, Nucleic Acids Research, 25: 2816-2822 (1997); Hobb, Jr., U.S.Pat. No. 4,997,928; Khanna et al., U.S. Pat. No. 4,318,846; and thelike. Preferably, D is a fluorescein or a fluorescein derivative.

In an embodiment illustrated in FIG. 2A, binding compounds comprise abiotinylated antibody (300) as a binding moiety. Molecular tags areattached to binding moiety (300) by way of avidin or streptavidin bridge(306). Preferably, in operation, binding moiety (300) is first reactedwith a target complex, after which avidin or streptavidin is added (304)to form antibody-biotin-avidin complex (305). To such complexes (305)are added (308) biotinylated molecular tags (310) to form bindingcompound (312).

In still another embodiment illustrated in FIG. 2B, binding compoundscomprise an antibody (314) derivatized with a multi-functional moiety(316) that contains multiple functional groups (318) that are reacted(320) molecular tag precursors to give a final binding compound havingmultiple molecular tags (322) attached. Exemplary multi-functionalmoieties include aminodextran, and like materials.

Once each of the binding compounds is separately derivatized by adifferent molecular tag, it is pooled with other binding compounds toform a plurality of binding compounds. Usually, each different kind ofbinding compound is present in a composition in the same proportion;however, proportions may be varied as a design choice so that one or asubset of particular binding compounds are present in greater or lowerproportion depending on the desirability or requirements for aparticular embodiment or assay. Factors that may affect such designchoices include, but are not limited to, antibody affinity and avidityfor a particular target, relative prevalence of a target, fluorescentcharacteristics of a detection moiety of a molecular tag, and the like.

B. Cleavage-Inducing Moiety Producing Active Species

A cleavage-inducing moiety, or cleaving agent, is a group that producesan active species that is capable of cleaving a cleavable linkage,preferably by oxidation. Preferably, the active species is a chemicalspecies that exhibits short-lived activity so that its cleavage-inducingeffects are only in the proximity of the site of its generation. Eitherthe active species is inherently short lived, so that it will not createsignificant background because beyond the proximity of its creation, ora scavenger is employed that efficiently scavenges the active species,so that it is not available to react with cleavable linkages beyond ashort distance from the site of its generation. Illustrative activespecies include singlet oxygen, hydrogen peroxide, NADH, and hydroxylradicals, phenoxy radical, superoxide, and the like. Illustrativequenchers for active species that cause oxidation include polyenes,carotenoids, vitamin E, vitamin C, amino acid-pyrrole N-conjugates oftyrosine, histidine, and glutathione, and the like, e.g. Beutner et al,Meth. Enzymol., 319: 226-241 (2000).

An important consideration in designing assays employing acleavage-inducing moiety and a cleavable linkage is that they not be sofar removed from one another when bound to a receptor complex that theactive species generated by the cleavage-inducing moiety cannotefficiently cleave the cleavable linkage. In one aspect, cleavablelinkages preferably are within 1000 nm, and preferably within 20-200 nm,of a bound cleavage-inducing moiety. More preferably, forphotosensitizer cleavage-inducing moieties generating singlet oxygen,cleavable linkages are within about 20-100 nm of a photosensitizer in areceptor complex. The range within which a cleavage-inducing moiety caneffectively cleave a cleavable linkage (that is, cleave enough moleculartag to generate a detectable signal) is referred to herein as its“effective proximity.” One of ordinary skill in the art recognizes thatthe effective proximity of a particular sensitizer may depend on thedetails of a particular assay design and may be determined or modifiedby routine experimentation.

A sensitizer is a compound that can be induced to generate a reactiveintermediate, or species, usually singlet oxygen. Preferably, asensitizer used in accordance with the invention is a photosensitizer.Other sensitizers included within the scope of the invention arecompounds that on excitation by heat, light, ionizing radiation, orchemical activation will release a molecule of singlet oxygen. The bestknown members of this class of compounds include the endoperoxides suchas 1,4-biscarboxyethyl-1,4-naphthalene endoperoxide,9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light bythese compounds releases singlet oxygen. Further sensitizers aredisclosed in the following references: Di Mascio et al, FEBS Lett., 355:287 (1994)(peroxidases and oxygenases); Kanofsky, J. Biol. Chem. 258:5991-5993 (1983)(lactoperoxidase); Pierlot et al, Meth. Enzymol., 319:3-20 (2000)(thermal lysis of endoperoxides); and the like. Attachment ofa binding agent to the cleavage-inducing moiety may be direct orindirect, covalent or non-covalent and can be accomplished by well-knowntechniques, commonly available in the literature. See, for example,“Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York (1978);Cuatrecasas, J. Biol. Chem., 245:3059 (1970).

As mentioned above, the preferred cleavage-inducing moiety in accordancewith the present invention is a photosensitizer that produces singletoxygen. As used herein, “photosensitizer” refers to a light-adsorbingmolecule that when activated by light converts molecular oxygen intosinglet oxygen. Photosensitizers may be attached directly or indirectly,via covalent or non-covalent linkages, to the binding agent of aclass-specific reagent. Guidance for constructing of such compositions,particularly for antibodies as binding agents, available in theliterature, e.g. in the fields of photodynamic therapy,immunodiagnostics, and the like. The following are exemplary references:Ullman, et al., Proc. Natl. Acad. Sci. USA 91, 5426-5430 (1994); Stronget al, Ann. New York Acad. Sci., 745: 297-320 (1994); Yarmush et al,Crit. Rev. Therapeutic Drug Carrier Syst., 10: 197-252 (1993); Pease etal, U.S. Pat. No. 5,709,994; Ullman et al, U.S. Pat. No. 5,340,716;Ullman et al, U.S. Pat. No. 6,251,581; McCapra, U.S. Pat. No. 5,516,636;and the like.

A large variety of light sources are available to photo-activatephotosensitizers to generate singlet oxygen. Both polychromatic andmonchromatic sources may be used as long as the source is sufficientlyintense to produce enough singlet oxygen in a practical time duration.The length of the irradiation is dependent on the nature of thephotosensitizer, the nature of the cleavable linkage, the power of thesource of irradiation, and its distance from the sample, and so forth.In general, the period for irradiation may be less than about amicrosecond to as long as about 10 minutes, usually in the range ofabout one millisecond to about 60 seconds. The intensity and length ofirradiation should be sufficient to excite at least about 0.1% of thephotosensitizer molecules, usually at least about 30% of thephotosensitizer molecules and preferably, substantially all of thephotosensitizer molecules. Exemplary light sources include, by way ofillustration and not limitation, lasers such as, e.g., helium-neonlasers, argon lasers, YAG lasers, He/Cd lasers, and ruby lasers;photodiodes; mercury, sodium and xenon vapor lamps; incandescent lampssuch as, e.g., tungsten and tungsten/halogen; flashlamps; and the like.By way of example, a photoactivation device disclosed in Bjornson et al,International patent publication WO 03/051669 is employed. Briefly, thephotoactivation device is an array of light emitting diodes (LEDs)mounted in housing that permits the simultaneous illumination of all thewells in a 96-well plate. A suitable LED for use in the presentinvention is a high power GaAIAs IR emitter, such as model OD-880Wmanufactured by OPTO DIODE CORP. (Newbury Park, Calif.).

Examples of photosensitizers that may be utilized in the presentinvention are those that have the above properties and are enumerated inthe following references: Singh and Ullman, U.S. Pat. No. 5,536,834; Liet al, U.S. Pat. No. 5,763,602; Martin et al, Methods Enzymol., 186:635-645 (1990); Yarmush et al, Crit. Rev. Therapeutic Drug CarrierSyst., 10: 197-252 (1993); Pease et al, U.S. Pat. No. 5,709,994; Ullmanet al, U.S. Pat. No. 5,340,716; Ullman et al, U.S. Pat. No. 6,251,581;McCapra, U.S. Pat. No. 5,516,636; Thetford, European patent publ.0484027; Sessler et al, SPIE, 1426: 318-329 (1991); Magda et al, U.S.Pat. No. 5,565,552; Roelant, U.S. Pat. No. 6,001,673; and the like.

As with sensitizers, in certain embodiments, a photosensitizer may beassociated with a solid phase support by being covalently ornon-covalently attached to the surface of the support or incorporatedinto the body of the support. In general, the photosensitizer isassociated with the support in an amount necessary to achieve thenecessary amount of singlet oxygen. Generally, the amount ofphotosensitizer is determined empirically.

In one embodiment, a photosensitizer is incorporated into a latexparticle to form photosensitizer beads, e.g. as disclosed by Pease etal., U.S. Pat. No. 5,709,994; Pollner, U.S. Pat. No. 6,346,384; andPease et al, PCT publication WO 01/84157. Alternatively, photosensitizerbeads may be prepared by covalently attaching a photosensitizer, such asrose bengal, to 0.5 micron latex beads by means of chloromethyl groupson the latex to provide an ester linking group, as described in J. Amer.Chem. Soc., 97: 3741 (1975). Use of such photosensitizer beads isillustrated in FIG. 2C, in which item labels (100)-(122) are describedin FIG. 1B. Reactions may be carried out, for example, in a conventional96-well or 384-well microtiter plate, or the like, having a filtermembrane that forms one wall, e.g. the bottom, of the wells that allowsreagents to be removed by the application of a vacuum. This allows theconvenient exchange of buffers, if the buffer required for specificbinding of binding compounds is different that the buffer required foreither singlet oxygen generation or separation. For example, in the caseof antibody-based binding compounds, a high salt buffer is required. Ifelectrophoretic separation of the released tags is employed, then betterperformance is achieved by exchanging the buffer for one that has alower salt concentration suitable for electrophoresis. In thisembodiment, instead of attaching a photosensitizer directly to a bindingcompound, such as an antibody, a cleaving probe comprises twocomponents: antibody (232) derivatized with a capture moiety, such asbiotin (indicated in FIG. 2C as “bio”) and photosensitizer bead (238)whose surface is derivatized with an agent (234) that specifically bindswith the capture moiety, such as avidin or streptavidin. Complexes (230)are then captured (236) by photosensitizer beads by way of the capturemoiety, such as streptavidin (234). Conveniently, if the pore diameterof the filter membrane is selected so that photosensitizer beads (238)cannot pass, then a buffer exchange also serves to remove unboundbinding compounds, which leads to an improved signal. After anappropriate buffer for separation has been added, if necessary,photosensitizer beads (238) are illuminated (240) so that singlet oxygenis generated (242) and molecular tags are released (244). Such releasedmolecular tags (346) are then separated to form separation profile (352)and dimers are quantified ratiometrically from peaks (348) and (350).Photosensitizer beads may be used in either homogeneous or heterogeneousassay formats.

Preferably, when analytes, such as cell surface receptors, are beingdetected or antigen in a fixed sample, a cleaving probe may comprise aprimary haptenated antibody and a secondary anti-hapten binding proteinderivatized with multiple photosensitizer molecules. A preferred primaryhaptenated antibody is a biotinylated antibody, and preferred secondaryanti-hapten binding proteins may be either an anti-biotin antibody orstreptavidin. Other combinations of such primary and secondary reagentsare well known in the art, e.g. Haugland, Handbook of Fluorescent Probesand Research Reagents, Ninth Edition (Molecular Probes, Eugene, Oreg.,2002). An exemplary combination of such reagents is illustrated in FIG.3. There binding compounds (366 and 368) having releasable tags (“mT₁”and “mT₂” in the Figure), and primary antibody (368) derivatized withbiotin (369) are specifically bound to different epitopes of receptordimer (362) in membrane (360). Biotin-specific binding protein (370),e.g. streptavidin, is attached to biotin (369) bringing multiplephotosensitizers (372) into effective proximity of binding compounds(366 and 368). Biotin-specific binding protein (370) may also be ananti-biotin antibody, and photosensitizers may be attached via freeamine group on the protein by conventional coupling chemistries, e.g.Hermanson (cited above). An exemplary photosensitizer for such use is anNHS ester of methylene blue prepared as disclosed in Shimadzu et al,European patent publication 0510688.

Assay Conditions

The following general discussion of methods and specific conditions andmaterials are by way of illustration and not limitation. One of ordinaryskill in the art will understand how the methods described herein can beadapted to other applications, particularly with using differentsamples, cell types and target complexes.

In conducting the methods of the invention, a combination of the assaycomponents is made, including the sample being tested, the bindingcompounds, and optionally the cleaving probe. Generally, assaycomponents may be combined in any order. In certain applications,however, the order of addition may be relevant. For example, one maywish to monitor competitive binding, such as in a quantitative assay. Orone may wish to monitor the stability of an assembled complex. In suchapplications, reactions may be assembled in stages, and may requireincubations before the complete mixture has been assembled, or beforethe cleaving reaction is initiated.

The amounts of each reagent are usually determined empirically. Theamount of sample used in an assay will be determined by the predictednumber of target complexes present and the means of separation anddetection used to monitor the signal of the assay. In general, theamounts of the binding compounds and the cleaving probe are provided inmolar excess relative to the expected amount of the target molecules inthe sample, generally at a molar excess of at least 1.5, more desirablyabout 10-fold excess, or more. In specific applications, theconcentration used may be higher or lower, depending on the affinity ofthe binding agents and the expected number of target molecules presenton a single cell. Where one is determining the effect of a chemicalcompound on formation of oligomeric cell surface complexes, the compoundmay be added to the cells prior to, simultaneously with, or afteraddition of the probes, depending on the effect being monitored.

The assay mixture is combined and incubated under conditions thatprovide for binding of the probes to the cell surface molecules, usuallyin an aqueous medium, generally at a physiological pH (comparable to thepH at which the cells are cultures), maintained by a buffer at aconcentration in the range of about 10 to 200 mM. Conventional buffersmay be used, as well as other conventional additives as necessary, suchas salts, growth medium, stabilizers, etc. Physiological and constanttemperatures are normally employed. Incubation temperatures normallyrange from about 4° to 70° C., usually from about 15° to 45° C., moreusually 25° to 37°.

After assembly of the assay mixture and incubation to allow the probesto bind to cell surface molecules, the mixture is treated to activatethe cleaving agent to cleave the tags from the binding compounds thatare within the effective proximity of the cleaving agent, releasing thecorresponding tag from the cell surface into solution. The nature ofthis treatment will depend on the mechanism of action of the cleavingagent. For example, where a photosensitizer is employed as the cleavingagent, activation of cleavage will comprise irradiation of the mixtureat the wavelength of light appropriate to the particular sensitizerused.

Following cleavage, the sample is then analyzed to determine theidentity of tags that have been released. Where an assay employing aplurality of binding compounds is employed, separation of the releasedtags will generally precede their detection. The methods for bothseparation and detection are determined in the process of designing thetags for the assay. A preferred mode of separation employselectrophoresis, in which the various tags are separated based on knowndifferences in their electrophoretic mobilities.

As mentioned above, in some embodiments, if the assay reactionconditions may interfere with the separation technique employed, it maybe necessary to remove, or exchange, the assay reaction buffer prior tocleavage and separation of the molecular tags. For example, assayconditions may include salt concentrations (e.g. required for specificbinding) that degrade separation performance when molecular tags areseparated on the basis of electrophoretic mobility. Thus, such high saltbuffers may be removed, e.g. prior to cleavage of molecular tags, andreplaced with another buffer suitable for electrophoretic separationthrough filtration, aspiration, dilution, or other means.

EXAMPLES Sources of Materials Used in Examples

Antibodies specific for Her receptors, adaptor molecules, andnormalization standards are obtained from commercial vendors, includingLabvision, Cell Signaling Technology, and BD Biosciences. All cell lineswere purchased from ATCC. All human snap-frozen tissue samples werepurchased from either William Bainbridge Genome Foundation (Seattle,Wash.) or Bio Research Support (Boca Raton, Fla.) and were approved byInstitutional Research Board (IRB) at the supplier.

The molecular tag-antibody conjugates used below are formed by reactingNHS esters of the molecular tag with a free amine on the indicatedantibody using conventional procedures. Molecular tags, identified belowby their “Pro_N” designations, are disclosed in the followingreferences: Singh et al, U.S. patent publications, 2003/017915 and2002/0013126, which are incorporated by reference. Briefly, bindingcompounds below are molecular tag-monoclonal antibody conjugates formedby reacting an NHS ester of a molecular tag with free amines of theantibodies in a conventional reaction.

Example 1 Simultaneous Measurement of Her2-Her3 Heterodimerization andErk1 Phosphorylation

In this example, an assay is described for providing a ratiometricmeasure of phosphorylated Erk1 and Her2-Her3 heterodimerization. Theassays are carried out as follows.

Sample Preparation:

-   -   1. Serum-starve breast cancer cell line culture (MCF-7)        overnight before use.    -   2. Stimulate cell lines with HRG in culture media for 10 minutes        at 37° C. Exemplary doses of HRG are 0, 0.032, 0.16, 0.8, 4, 20,        100 nM for MCF-7 cells.    -   3. Aspirate culture media, transfer onto ice, and add lysis        buffer (described below) to lyse cells in situ.    -   4. Scrape and transfer lysate to microfuge tube. Incubate on ice        for 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min.    -   5. Collect supernatants as lysates and aliquot for storage at        −80° C. until use.

Lysis Buffer (Made Fresh and Stored on Ice):

Final ul Stock 1% Triton X-100 1000 10% 20 mM Tris-HCl (pH 7.5) 200 1 M100 mM NaCl 200 5 M 50 mM NaF 500 1 M 50 mM Na beta-glycerophosphate1000 0.5 M 1 mM Na₃ VO₄ 100 0.1 M 5 mM EDTA 100 0.5 M 10 ug/ml pepstatin100 1 mg/ml 1 tablet (per 10 ml) Roche Complete protease N/A N/Ainhibitor (#1836170) Water 6500 N/A 10 ml Total

The total assay volume is 40 ul. The lysate volume is adjusted to 10 ulwith lysis buffer. The antibodies are diluted in lysis buffer up to 20ul. Typically ˜5000 to 500,000 cell-equivalent of lysates is used perreaction.

Procedure: Working concentrations of pre-mixed antibodies prior toadding into reaction:

eTag1_anti-Erk1 (epitope 1) at 10 nM

eTag2_anti-phospho-Erk1 at 10 nM

Biotin_anti-Erk1 (epitope 2) at 20 nM

eTag3_anti-Her2 at 10 nM

eTag4_anti-phospho-Her2

Biotin_anti-Her3 at 20 nM

Universal Standard US-1 at 700 nM

-   -   [The Universal Standard US-1 is BSA conjugated with biotin and        molecular tag Pro8, which is used to normalize the amount of        streptavidin-photosensitizer beads in an assay].    -   The molecular tags are attached directly to antibodies by        reacting an NHS-ester of a molecular tag precursor (see FIGS.        15A-15J in U.S. patent publication 2003/0013126 A1, which is        incorporated herein by reference) with free amines on the        antibodies using conventional techniques, e.g. Hermanson (cited        above).    -   1. To assay 96-well filter plate (Millipore MAGVN2250), add 20        ul antibody mix to 10 ul lysate and incubate for 1 hour at 4° C.    -   2. Add 10 ul streptavidin-derivatized cleaving probe (final 4        ug/well) to assay well and incubate for 40 min.    -   3. Add 200 ul wash buffer and apply vacuum to empty.    -   4. Add 30 ul illumination buffer and illuminate.    -   5. Transfer 10 ul of each reaction to CE assay plate for        analysis.

Data Analysis:

-   -   1. Normalize relative fluorescence units (RFU) signal of each        molecular tag against that of internal Universal Standard US-1.    -   2. Subtract RFU of “no lysate” background control from        corresponding normalized eTag reporter signals.

Example 2 Analysis of Cell Lysates for Her-2 Heterodimerization andReceptor Phosphorylation

In this example, Her1-Her2 and Her2-Her3 heterodimers andphosphorylation states are measured in cell lysates from several celllines after treatment with various concentrations of epidermal growthfactor (EGF) and heregulin (HRG). Measurements are made using threebinding compounds and a cleaving probe as described below.

Sample Preparation:

-   -   1. Serum-starve breast cancer cell line culture overnight before        use.    -   2. Stimulate cell lines with EGF and/or HRG in culture media for        10 minutes at 37° C. Exemplary doses of EGF/HRG are 0, 0.032,        0.16, 0.8, 4, 20, 100 nM for all cell lines (e.g. MCF-7, T47D,        SKBR-3) except BT20 for which the maximal dose is increased to        500 nM because saturation is not achieved with 100 nM EGF.    -   3. Aspirate culture media, transfer onto ice, and add lysis        buffer to lyse cells in situ.    -   4. Scrape and transfer lysate to microfuge tube. Incubate on ice        for 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min.        (Centrifugation is optional.)    -   5. Collect supernatants as lysates and aliquot for storage at        −80° C. until use.

Assay:

Assay design: As illustrated diagrammatically in FIG. 4A, Her2-Her3heterodimers (900) are quantified ratiometrically based on the bindingof cleaving probe (902) and binding compounds (904), (906), and (908). Aphotosensitizer indicated by “PS” is attached to cleaving probe (902)via an avidin-biotin linkage, and binding compounds (904), (906), and(908) are labeled with molecular tags Pro 14, Pro10, and Pro11,respectively. Binding compound (904) is specific for a phosphorylationsite on Her3.The total assay volume is 40 ul. The lysate volume is adjusted to 30 ulwith lysis buffer. The antibodies are diluted in lysis buffer up to 10ul. Typically ˜5000 to 15000 cell-equivalent of lysates is used perreaction. The detection limit is ˜1000 cell-equivalent of lysates.Procedure: Final concentrations of pre-mixed binding compounds (i.e.molecular tag- or biotin-antibody conjugates) in reaction:Pro4_anti-Her-2: 0.1 ug/mlPro10_anti-Her-1: 0.05-0.1 ug/mlPro11_anti-Her-3: 0.1 ug/mlPro2_anti-phospho-Tyr: 0.1 ug/mlBiotin_anti-Her-2: 1-2 ug/ml

-   -   6. To assay 96-well, add 10 ul antibody mix to 30 ul lysate and        incubate for 1 hour at RT.    -   7. Add 2 ul streptavidin-derivatized cleaving probe (final 2        ug/well) to assay well and incubate for 45 min.    -   8. Add 150 ul of PBS with 1% BSA to 96-well filter plate        (Millipore MAGVN2250) and incubate for 1 hr at RT for blocking.    -   9. Empty filter plate by vacuum suction. Transfer assay        reactions to filter plate and apply vacuum to empty.    -   10. Add 200 ul wash buffer and apply vacuum to empty. Repeat one        time.    -   11. Add 200 ul illumination buffer and apply vacuum to empty.        Repeat one time.    -   12. Add 30 ul illumination buffer and illuminate for 20 min.    -   13. Transfer 10 ul of each reaction to CE assay plate for        analysis using an ABI3100 CE instrument with a 22 cm capillary        (injection conditions: 5 kV, 75 sec, 30° C.; run conditions: 600        sec, 30° C.).

Assay Buffers are as Follows: Lysis Buffer (Made Fresh and Stored onIce)

Final ul Stock 1% Triton X-100 1000 10% 20 mM Tris-HCl (pH 7.5) 200 1 M100 mM NaCl 200 5 M 50 mM NaF 500 1 M 50 mM Na beta-glycerophosphate1000 0.5 M 1 mM Na₃ VO₄ 100 0.1 M 5 mM EDTA 100 0.5 M 10 ug/ml pepstatin100 1 mg/ml 1 tablet (per 10 ml) Roche Complete protease N/A N/Ainhibitor (#1836170) Water 6500 N/A 10 ml Total

Wash Buffer (Stored at 4° C.)

Final ml Stock 1% NP-40 50 10% 1x PBS 50 10x 150 mM NaCl 15   5 M 5 mMEDTA 5 0.5 M Water 380 N/A 500 ml Total

Illumination Buffer:

Final ul Stock 0.005x PBS 50  1x CE std 3 100x 10 mM Tris-HCl (pH 8.0)0.1M 10 pM A160 1 nM 10 pM A315 1 nM 10 pM HABA 1 nM Water 10,000 NA 10ml Total

Data Analysis:

-   -   3. Normalize relative fluorescence units (RFU) signal of each        molecular tag against CE reference standard A315 (a        fluorescein-derivatized deoxyadenosine monophosphate that has        known peak position relative to molecular tags from the assay        upon electrophoretic separation).    -   4. Subtract RFU of “no lysate” background control from        corresponding molecular tag signals.    -   5. Report heterodimerization for Her-1 or Her-3 as the        corresponding RFU ratiometric to RFU from Pro4_anti-Her-2 from        assay wells using biotin-anti-Her-2.    -   6. Report receptor phosphorylation for Her-1,2,3 as RFU from        Pro2_PT100 anti-phospho-Tyr ratiometric to RFU from        Pro4_anti-Her-2 from assay wells using biotin-anti-Her-2.        Results of the assays are illustrated in FIGS. 4B-4H. FIG. 4B        shows the quantity of Her1-Her2 heterodimers increases on MCF-7        cells with increasing concentrations of EGF, while the quantity        of the same dimer show essentially no change with increasing        concentrations of HRG. FIG. 4C shows the opposite result for        Her2-Her3 heterodimers. That is, the quantity of Her2-Her3        heterodimers increases on MCF-7 cells with increasing        concentrations of HRG, while the quantity of the same dimer show        essentially no change with increasing concentrations of EGF.        FIGS. 4D and 4E show the quantity of Her1-Her2 heterodimers        increases on SKBR-3 cells and BT-20 cells, respectively, with        increasing concentrations of EGF.

Example 3 Analysis of Tissue Lysates for Her2 Heterodimerization andReceptor Phosphorylation

In this example, Her1-Her2 and Her2-Her3 heterodimers andphosphorylation states are measured in tissue lysates from human breastcancer specimens.

Sample Preparation:

-   -   1. Snap frozen tissues are mechanically disrupted at the frozen        state by cutting.    -   2. Transfer tissues to microfuge tube and add 3× tissue volumes        of lysis buffer (from appendix I) followed by vortexing to        disperse tissues in buffer.    -   3. Incubate on ice for 30 min with intermittent vortexing to        mix.    -   4. Centrifuge at 14,000 rpm, 4° C., for 20 min.    -   5. Collect supernatants as lysates and determine total protein        concentration with BCA assay (Pierce) using a small aliquot.    -   6. Aliquot the rest for storage at −80° C. until use.

Assay Design:

-   -   1. The total assay volume is 40 ul.    -   2. The lysates are tested in serial titration series of 40, 20,        10, 5, 2.5, 1.25, 0.63, 0.31 ug total-equivalents and the volume        is adjusted to 30 ul with lysis buffer. Data from the titration        series confirm the specificity of the dimerization or        phosphorylation signals.    -   3. A universal antibody mix comprising all eTag-antibodies        diluted in lysis buffer is used at the following concentrations.    -   4. Individual biotin-antibody for each receptor is added        separately to the reactions.    -   5. Three eTag assays are conducted with each tissue lysate, each        using a different biotin-antibody corresponding to specific        receptor dimerization to be measured.    -   6. Expression level of each receptor is determined from        different assay containing the biotin-antibody specific to the        receptor.    -   7. Dimerization and phosphorylation signals are determined        ratiometrically only in the assay containing the        biotin-anti-Her-2.        Assay controls: MCF-10A and MCF-7 cell lines are used as        qualitative negative and positive controls, respectively. Cell        lines are either unstimulated or stimulated with 100 nM EGF or        100 nM HRG. Lysis buffer is included as a background control        when replacing the tissue samples.

Final Concentrations of Pre-Mixed Antibodies in Reactions: UniversalAntibody Mix:

Pro4_anti-Her-2: 0.1 ug/ml

Pro10_anti-Her-1: 0.05 ug/ml

Pro11_anti-Her-3: 0.1 ug/ml

Pro2_anti-phospho-Tyr: 0.01 ug/ml

Individual Biotin Antibody:

Biotin_anti-Her-1: 2 ug/ml

Biotin_anti-Her-2: 2 ug/ml

Biotin_anti-Her-3: 2 ug/ml

Procedure:

-   -   1. Prepare antibody reaction mix by adding biotin antibody to        universal antibody mix.    -   2. To assay 96-well, add 10 ul universal reaction mix to 30 ul        lysate and incubate for 1 hour at RT.    -   3. Add 2 ul streptavidin-derivatized cleaving probe (final 2        ug/well) to assay well and incubate for 45 min.    -   4. Add 150 ul of PBS with 1% BSA to 96-well filter plate        (Millipore MAGVN2250) and incubate for 1 hr at RT for blocking.    -   5. Empty filter plate by vacuum suction. Transfer assay        reactions to filter plate and apply vacuum to empty.    -   6. Add 200 ul wash buffer and apply vacuum to empty. Repeat one        time.    -   7. Add 200 ul illumination buffer and apply vacuum to empty.        Repeat one time.    -   8. Add 30 ul illumination buffer and illuminate for 20 min.    -   9. Transfer 10 ul of each reaction to CE assay plate for        analysis using ABI3100 capillary electrophoresis instrument with        a 22 cm capillary (injection conditions: 5 kV, 75 sec, 30° C.;        run conditions: 600 sec, 30° C.)

Data Analysis:

-   -   1. Normalize RFU signal of each molecular tag against CE        reference standard A315.    -   2. Determine the cut-off values of RFU (each for dimerization or        phosphorylation) below which ratios are not calculated because        the signals are too low to be reliable. Below the cut-off        values, the RFU signals are not titratable in the series of        lysate dilution tested. The values can be determined with a        large set of normal tissues where dimerization and        phosphorylation signals are expected to be absent or at the        lowest. These values also represent the basal level of        dimerization or phosphorylation on the normal tissues to which        tumor tissues will be compared.    -   3. For the minority of normal tissues, if present, with RFU        values above the cut-off, determine the individual RFU level and        ratiometric readouts of Her-1 or Her-3 heterodimerization or        phosphorylation peaks detected. These samples represent outliers        that should be used as matched donor controls for the        corresponding tumor tissue samples while scoring.    -   4. For all tumor samples showing titratable RFU signals, use the        lowest signal of each of Her-1, Her-2, Her-3, or phosphorylation        from the tissue lysate titration series as the background.        Subtract this background from the molecular tag signals of the        high dose lysates (e.g. 40 ug) to yield the specific RFU        signals. If there is no signal dose response in the titration        series, all signals (which are usually very low) are considered        background and no specific signals can be used for ratiometric        analysis.    -   5. Report heterodimerization for Her-1 or Her-3 as the        corresponding specific RFU ratiometric to the specific RFU from        Pro4_anti-Her-2. If no specific RFU is obtained, the        dimerization is negative.    -   6. Report receptor phosphorylation for Her-1,2,3 as specific RFU        from Pro2_anti-phospho-Tyr ratiometric to the specific RFU from        Pro4_anti-Her-2. If no specific RFU is obtained, the        phosphorylation is negative.        In FIGS. 5A-5C data shown are representative of multiple        patients' breast tissue samples tested with assays of the        invention. The clinical Her-2 status from immunohistochemistry        (DAKO Herceptest) of 9 out of 10 tumor samples was negative,        indicative of either undetectable Her-2 staining, or staining of        less than 10% of the tumor cells, or a faint and barely        perceptible staining on part of the cell membrane of more than        10% tumor cells. The assays of the invention determined the        expression of Her-1, Her-2, and Her-3 on both normal and tumor        tissues. The heterodimerization of Her1 and the Her2 and of Her2        and Her3 was detected only in tumor tissues but not in any        normal tissues.

Example 4 Analysis of Cell Lysates for Her1 or Her2 Homodimerization andReceptor Phosphorylation

Sample preparation was carried out essentially as described in Example2. Her1 homodimerization was induced by treating the cell lines with EGFor TGFα. For homodimerization of Her2 which does not have a ligand,unstimulated SKBR-3 or MDA-MD-453 cells that overexpress Her2 arecompared to unstimulated MCF-7 cells that express a low level of Her2.

Assay design: A monoclonal antibody specific to the receptor isseparately conjugated with either a molecular tag or biotin (that isthen linked to a photosensitizer via an avidin bridge), so that thecleaving probe and a binding compound compete to bind to the sameepitope in this example. Another binding compound is used that consistsof a second antibody recognizing an overlapping epitope on the receptor,so that a ratiometric signal can be generated as a measure ofhomodimerization. The signal derived from the second antibody alsoprovides a measure of the total amount of receptor in a sample. Thetotal amount of receptor is determined in a separate assay well.Receptor phosphorylation can be quantified together with eitherhomodimerization or total receptor amount.

Procedure: The assay volume is 40 ul and the general procedure issimilar to that of Example 2. Two assay wells, A and B, are set up foreach sample to quantify homodimerization and total amount of receptorseparately.

For Quantification of Her1-Her1 Homodimers: Final Concentrations inAntibody Mix in Assay Well A:

Pro 12_anti-Her-1: 0.05-0.1 ug/ml

Biotin_anti-Her-1: 1-2 ug/ml

Final Concentrations in Antibody Mix in Assay Well B:

Pro10_anti-Her-1: 0.05-0.1 ug/ml

Pro2_anti-phospho-Tyr: 0.1 ug/ml

Biotin_anti-Her-1: 1-2 ug/ml

For Quantification of Her2-Her2 Homodimers: Final Concentrations inAntibody Mix in Assay Well A:

Pro4_anti-Her-1: 0.05-0.1 ug/ml

Biotin_anti-Her-1: 1-2 ug/ml

Final Concentrations in Antibody Mix in Assay Well B:

Pro4_anti-Her-1: 0.05-0.1 ug/ml

Pro2_anti-phospho-Tyr: 0.1 ug/ml

Biotin_anti-Her-1: 1-2 ug/ml

Data Analysis:

-   -   1. Normalize RFU signal of each molecular tag against CE        reference standard A315.    -   2. Subtract RFU of “no lysate” background control from        corresponding molecular tag signals.    -   3. Report homodimerization for Her-1 or Her-2 as the        corresponding normalized RFU from assay well A as ratiometric to        normalized RFU of total receptor amount from the corresponding        assay well B.    -   4. Report receptor phosphorylation for Her-1 or Her-2 homodimer        as normalized RFU from Pro2_PT 100 anti-phospho-Tyr from assay        well B as ratiometric to normalized RFU from total receptor        amount from the same assay well B.        Results of the assays are illustrated in FIGS. 6A-6B and FIG. 7.        FIG. 6A shows that the quantity of Her1-Her1 homodimers on BT-20        cells increases with increasing concentration of EGF. FIG. 6B        shows that the quantity of Her1 phosphorylation in BT-20 cells        increases with increasing EGF concentration. The detection of        Her2-Her2 homodimers was demonstrated by comparison of signals        from SKBR-3 cells expressing Her2 with signals from MCF-7 cells        that express reduced level of Her2 on the cell surface. As shown        in the charts of FIG. 7, no specific titratable Her2-Her2        homodimer signals were detected with MCF-7 cells whereas        Her2-Her2 homodimer signals from SKBR-3 cells were clearly above        the signals from MCF-7 cells

Example 5 Analysis of Cell Lysates for Her1-Her3 Heterodimerization andReceptor Phosphorylation Samples are Prepared as Follows:

-   -   1. Serum-starve breast cancer cell line culture overnight before        use.    -   2. Stimulate cell lines with HRG in culture media for 10 minutes        at 37° C. Exemplary doses of HRG are 0, 0.032, 0.16, 0.8, 4, 20,        100 nM for T47D cells.    -   3. Aspirate culture media, transfer onto ice, and add lysis        buffer to lyse cells in situ.    -   4. Scrape and transfer lysate to microfuge tube. Incubate on ice        for 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min.        (Centrifugation is optional.)    -   5. Collect supernatants as lysates and aliquot for storage at        −80° C. until use.        Assay design: The total assay volume is 40 ul. The lysate volume        is adjusted to 30 ul with lysis buffer. The antibodies are        diluted in lysis buffer up to 5 ul. Typically ˜5000 to 50000        cell-equivalent of lysates is used per reaction. Final        concentrations of pre-mixed antibodies in reaction:

Pro10_anti-Her-1: 0.05-0.1 ug/ml

Pro11_anti-Her-3: 0.1 ug/ml

Pro2_anti-phospho-Tyr: 0.1 ug/ml

Biotin_anti-Her-3: 1-2 ug/ml

-   -   1. To assay 96-well, add 5 ul antibody mix to 30 ul lysate and        incubate for 1 hour at RT.    -   2. Add 5 ul streptavidin-derivatized molecular scissor (final 4        ug/well) to assay well and incubate for 45 min.    -   3. Add 150 ul of PBS with 1% BSA to 96-well filter plate        (Millipore MAGVN2250) and incubate for 1 hr at RT for blocking.    -   4. Empty filter plate by vacuum suction. Transfer assay        reactions to filter plate and apply vacuum to empty.    -   5. Add 200 ul wash buffer and apply vacuum to empty. Repeat one        time.    -   6. Add 200 ul illumination buffer and apply vacuum to empty.        Repeat one time.    -   7. Add 30 ul illumination buffer and illuminate for 20 Min.    -   8. Transfer 10 ul of each reaction to CE assay plate for        analysis using ABI3100 capillary electrophoresis instrument with        a 22 cm capillary (injection conditions: 5 kV, 425 sec, 30° C.;        run conditions: 600 sec, 30° C.).    -   Data Analysis:    -   1. Normalize RFU signal of each eTag reporter against CE        reference standard A315.    -   2. Subtract RFU of “no lysate” background control from        corresponding eTag reporter signals.    -   3. Report heterodimerization as the Her-1 derived Pro10 RFU        ratiometric to Pro11 RFU from anti-Her-3.    -   4. Report receptor phosphorylation for Her-1/3 as RFU from        Pro2_PT100 anti-phospho-Tyr ratiometric to RFU from        Pro11_anti-Her-3 from assay wells using biotin-anti-Her-3.        Results of the assay are illustrated in FIGS. 8A and 8B. The        data show that both Her1-Her3 heterodimerization and dimer        phosphorylation increase with increasing concentrations of HRG.

Example 6 Increase in Her1-Her3 Receptor Dimer Expression in Cancer CellLines in Response to Increase in Epidermal Growth Factor

In this example, Her1-Her3 heterodimers are measured in cell lysatesfrom cancer cell lines 22Rv1 and A549 after treatment with variousconcentrations of epidermal growth factor (EGF). Measurements are madeusing three binding compounds and a cleaving probe as described below.

Sample Preparation:

-   -   1. Serum-starve breast cancer cell line culture overnight before        use.    -   2. Stimulate cell lines with EGF in culture media for 10 minutes        at 37° C. Exemplary doses of EGF applied to both cell lines        varied between 0-100 nM.    -   3. Aspirate culture media, transfer onto ice, and add lysis        buffer to lyse cells in situ.    -   4. Scrape and transfer lysate to microfuge tube. Incubate on ice        for 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min.        (Centrifugation is optional.) Determine protein concentration.    -   5. Collect supernatants as lysates and aliquot for storage at        −80° C. until use.        The assay design is essentially the same as illustrated in FIG.        4A, with the following exceptions: binding compounds (904),        (906), and (908) are labeled with molecular tags Pro10, Pro 11,        and Pro 2, respectively. The total assay volume is 40 ul. The        lysate volume is adjusted to 30 ul with lysis buffer. The        antibodies are diluted in lysis buffer up to 5 ul. Typically        ˜5000 to 15000 cell-equivalent of lysates is used per reaction.        The detection limit is ˜1000 cell-equivalent of lysates.        Procedure: Final concentrations of pre-mixed binding compounds        (i.e. molecular tag- or biotin-antibody conjugates) in reaction:

Pro10_anti-Her-1: 0.05-0.1 ug/ml

Pro11_anti-Her-3: 0.1 ug/ml

Pro2_anti-phospho-Tyr: 0.1 to 0.2 ug/ml

Biotin_anti-Her-3: 1-2 ug/ml

-   -   1. To assay 96-well, add 5 ul antibody mix to 30 ul lysate and        incubate for 1 hour at RT.    -   2. Add 5 ul streptavidin-derivatized cleaving probe (final 4        ug/well) to assay well and incubate for 45 min.    -   3. Add 150 ul of PBS with 1% BSA to 96-well filter plate        (Millipore MAGVN2250) and incubate for 1 hr at RT for blocking.    -   4. Empty filter plate by vacuum suction. Transfer assay        reactions to filter plate and apply vacuum to empty.    -   5. Add 200 ul wash buffer and apply vacuum to empty. Repeat one        time.    -   6. Add 200 ul illumination buffer and apply vacuum to empty.        Repeat one time.    -   7. Add 30 ul illumination buffer and illuminate for 20 min.    -   8. Transfer 10 ul of each reaction to CE assay plate for        analysis using an ABI3100 CE instrument with a 22 cm capillary        (injection conditions: 5 kV, 70 sec, 30° C.; run conditions: 425        sec, 30° C.).

Assay Buffers are as Follows: Lysis Buffer (Made Fresh and Stored onIce)

Final ul Stock 1% Triton X-100 1000 10% 20 mM Tris-HCI (pH 7.5) 500 1 M100 mM NaCl 200 5 M 50 mM NaF 500 1 M 50 mM Na beta-glycerophosphate 5001.0 M 1 mM Na₃ VO₄ 100 0.1 M 5 mM EDTA 100 0.5 M 10 ug/ml pepstatin 1001 mg/ml 1 tablet (per 10 ml) Roche Complete protease N/A N/A inhibitor(#1836170) Water 7 ml N/A 10 ml TotalWash buffer (stored at 4° C.): 0.5% Triton X100 in 1×PBS.

Illumination Buffer:

Final ul Stock 0.005x PBS 50   1x CE std 1 (A27, ACLARA Biosciences, 45000x Inc., Mountain View, CA) CE std 2 (fluorescein) 4 5000x Water 9942N/A 10 ml Total

Data Analysis:

-   -   1. Normalize relative fluorescence units (RFU) signal of each        molecular tag against CE reference standard 2.    -   2. Subtract RFU of “no lysate” background control from        corresponding molecular tag signals.    -   3. Report heterodimerization for Her-1 as the corresponding RFU        ratiometric to RFU from Pro11_anti-Her-3 from assay wells using        biotin-anti-Her-3.    -   4. Report receptor phosphorylation for Her-1,2,3 as RFU from        Pro2_PT 100 anti-phospho-Tyr ratiometric to RFU from        Pro11_anti-Her-3 from assay wells using biotin-anti-Her-3 (data        not shown).        FIGS. 9A and 9B show the increases in the numbers of Her1-Her3        heterodimers on 22Rv1 and A549 cells, respectively, with        increasing concentrations of EGF.

Example 7 Occurrence of IGF-1R Heterodimers with Her1, Her2, and Her3 inBreast Tumor Tissue Lysates

In this example, cells from 12 different human breast tumor tissues wereassayed for the presence of Her1-IGF-1R, Her2-IGF-1R, and Her3-IGF-1Rdimers using assays essentially the same as that illustrated in FIG. 4A.Sample Preparation was carried out as follows:

-   -   1. Snap frozen tissues are mechanically disrupted at the frozen        state by cutting.    -   2. Transfer tissues to microfuge tube and add 3× tissue volumes        of lysis buffer followed by vortexing to disperse tissues in        buffer.    -   3. Incubate on ice for 30 min with intermittent vortexing to        mix.    -   4. Centrifuge at 14,000 rpm, 4° C., for 20 min.    -   5. Collect supernatants as lysates and determine total protein        concentration with BCA assay (Pierce) using a small aliquot.    -   6. Aliquot the rest for storage at −80° C. until use.        The assay was set up as follows.    -   1. The total assay volume is 40 ul.    -   2. The lysates are tested in serial titration series of 40, 20,        10, 5, 2.5, 1.25, 0.63, 0.31 ug total-equivalents and the volume        is adjusted to 30 ul with lysis buffer. Data from the titration        series confirm the specificity of the dimerization.    -   3. A universal antibody mix comprising of all binding compounds        and biotin antibody diluted in lysis buffer is used at        concentrations given below.

Final Concentrations of Pre-Mixed Antibodies in Reactions:

Pro10_anti-Her-2: 0.1 ug/ml

Pro14_anti-Her-1: 0.1 ug/ml

Pro11_anti-Her-3: 0.1 ug/ml

Pro7_anti-IGF-1R: 0.1 ug/ml

Pro2_anti-phospho-Tyr: 0.2 ug/ml

Biotin_anti-Her-2: 2 ug/ml

Procedure:

-   -   1. To assay 96-wells, add 5 ul universal reaction mix to 30 ul        lysate and incubate for 1 hour at RT.    -   2. Add 5 ul strepatvidin-derivatized molecular scissor (final 4        ug/well) to assay well and 5 incubate for 45 min.    -   3. Add 150 ul of PBS with 1% BSA to 96-well filter plate        (Millipore MAGVN2250) and incubate for 1 hr at RT for blocking.    -   4. Empty filter plate by vacuum suction. Transfer assay        reactions to filter plate and apply vacuum to empty.    -   5. Add 200 ul wash buffer and apply vacuum to empty. Repeat one        time.    -   6. Add 200 ul illumination buffer and apply vacuum to empty.        Repeat one time.    -   7. Add 30 ul illumination buffer and illuminate for 20 min.    -   8. Transfer 10 ul of each reaction to CE assay plate for        analysis using: (i) CE equipment: ABI3100, 22 cm capillary, (ii)        CE injection conditions: 5 kV, 70 sec, 30° C., and (iii) CE run        conditions: 425 sec, 30° C.

Data Analysis:

-   -   1. Normalize RFU signal of each molecular tag against CE        reference standard 1.    -   2. Look for titratable signals for each molecular tag. Signals        that do not titrate are assumed to be non-specific signals and        are not used for data interpretation. A cut off value is        determined based on the values from a large set of normal        tissues where dimerization signals are expected to be absent or        at the lowest. These values also represent the basal level of        dimerization on the normal tissues to which tumor tissues are        compared.    -   3. Heterodimerization is reported for IGF-1R with Her-1 or Her-2        or Her-3 as the corresponding specific RFU.        Two out of the twelve breast tumors assayed expressed        Her1-IGF-1R, Her2-IGF-1R, and Her3-IGF-1R heterodimers, as shown        in FIGS. 10A-C. The lines in each figure panel shows the trend        between receptor heterodimer quantity measured and amount of        lysate assayed for the two breast tumor samples that were        positive for the indicated heterodimers.

Example 8 Measurement of Receptor Dimers in Formalin Fixed ParaffinEmbedded Tissue Samples

In this example, model fixed tissues made from pelleted cell lines wereassayed for the presence of Her receptor dimers. The assay design forheterodimers was essentially the same as that described in FIG. 4A, withexceptions as noted below. That is, four components are employed: (i) acleaving probe comprising a biotinylated monoclonal antibody conjugatedto a cleavage-inducing moiety (in this example, aphotosensitizer-derivatized streptavidin, as illustrated in FIG. 3E) andspecific for one of the receptors of the dimer, (ii) a monoclonalantibody derivatized with a first molecular tag and specific for thesame receptor as the cleaving probe, (iii) a monoclonal antibodyderivatized with a second molecular tag and specific for the receptoropposite to that the cleaving probe is specific for, and (iv) amonoclonal antibody derivatized with a third molecular tag and specificfor an intracellular phosphorylated tyrosine. The assay design forhomodimers was essentially the same as that described in FIG. 1D, withexceptions as noted below.

In each case, model fixed tissues were prepared as follows: cells grownon tissue culture plates were stimulated with either EGF or HRG asdescribed in the prior examples, after which they were washed andremoved by scrapping. The removed cells were centrifuged to form apellet, after which formalin was added and the mixture was incubatedovernight at 4° C. The fixed pellet was embedded in paraffin using aMiles Tissue Tek III Embedding Center, after which 10 μm tissue sectionswere sliced from the pellet using a microtome (Leica model 2145). Tissuesections were placed on positively charged glass microscope slides(usually multiple tissue sections per slide) and baked for 1 hr at 60°C.

Tissue sections on the slides were assayed as follows: Tissue sectionson a slide were de-waxed with EZ-Dewax reagent (Biogenex, San Ramon,Calif.) using the manufacturer's recommended protocol. Briefly, 500 μLEZ-Dewax was added to each tissue section and the sections wereincubated at RT for 5 min, after which the slide was washed with 70%EtOH. This step was repeated and the slide was finally rinsed withdeionized water, after which the slide was incubated in water at RT for20 min. The slide was then immersed into a 1× Antigen Retrieval solution(Biogenesis, Brentwood, N.H.) at pH 10, after which it was heated for 15min in a microwave oven (5 min at high power setting followed by 10 minat a low power setting). After cooling to RT (about 45 min), the slidewas placed in a water bath for 5 min, then dried. Tissue sections on thedried slide were circled with a hydrophobic wax pen to create regionscapable of containing reagents placed on the tissue sections (asillustrated in FIGS. 3H-3I), after which the slide was washed threetimes in 1× Perm/Wash (BD Biosciences). To each section 50-100 μLblocking buffer was added, and the slide was placed in a coveredhumidified box containing deionized water for 2 hr at 4° C., after whichthe blocking buffer was removed from each section by suction. (Blockingbuffer is 1× Perm/Wash solution with protease inhibitors (Roche),phosphatase inhibitors (sodium floride, sodium vanadate, β-glycerolphosphate), and 10% mouse serum). To each section 40-50 μL of antibodymix containing binding compounds and cleaving probe was added (each at 5μg/mL, except that biotin-Ab5 (anti-Her1) was at 10 μg/mL in theHer1-Her2 assay), and the slide was placed in a humidified box overnightat 4° C. The sections were then washed three times with 100 μL Perm/Washcontaining protease and phosphatase inhibitors, after which 50 μL ofphotosensitizer in 1× Perm/Wash solution (containing protease andphosphatase inhibitors) was added. The slide was then incubated for1-1.5 hr at 4° C. in the dark in a humidified box, after which thephotosensitizer was removed by suction while keeping the slide in thedark. While remaining in the dark, the slide was then immersed in0.01×PBS and incubated on ice for 1 hr. The slide was remove from thePBS, dried, and to each section, 40-50 μL 0.01×PBS with 2 pM fluoresceinwas added, after which it was illuminated with a high power laser diode(GaAIAs IR emitter, model OD-880W, OPTO DIODE CORP, Newbury Park,Calif.) for 1 hr. The fluorescein acts as a standard to assist incorrelating peaks in an electropherogram with molecular tags. Afterillumination, the solution covering each tissue section was mixed bygentle pipeting and transferred to a CE plate for analysis on an AppliedBiosystems (Foster City, Calif.) model 3100 capillary electrophoresisinstrument.

FIG. 11A shows data from analysis of Her1-Her1 homodimers and receptorphosphorylation in sections from fixed pellets of breast adenocarcinomacell line, MDA-MB-468 (ATCC accession no. HTB-132), prepared from eithernon-stimulated cells or cells stimulated with 100 nM EGF. Biotinylatedanti-Her1 monoclonal antibody (Labvision) at 2 μg/mL was use as theprimary antibody of the cleaving probe (for cleavage methylene-bluederivatized streptavidin (described above) was attached through thebiotin). Pro10-derivatized anti-Her1 monoclonal antibody (Labvision) at2 μg/mL was used to measure homodimerized Her1. Pro1-derivatizedanti-Her1 monoclonal antibody (Labvision) at 0.8 μg/mL was used tomeasure total Her1. Unlabeled antibody Ab-5 was also included in thereactions at 3.2 μg/mL. Pro2-derivatized monoclonal antibody(anti-phosphorylated-Tyr, Cell Signaling) at 0.5 μg/mL was used tomeasure intracellular phosphorylation. The data from fixed tissuemeasurements confirm and are consistent with measurements on celllysates that show increases in Her1-Her1 homodimer expression andintracellular phosphoryation due to EGF stimulation.

FIG. 11B shows data from analysis of Her2-Her2 homodimers and receptorphosphorylation in sections from fixed pellets of breast cancer celllines MCF-7 and SKBR-3. All monoclonal antibodies used as cleavingprobes or binding compounds were used at concentrations of 5 μg/mL. Inorder to generate better cleavage, in this assay two cleaving probeswere employed, one directed to an extracellular antigenic determinant ofHer2 and one directed to an intracellular antigenic determinant of Her2.The data from fixed tissue measurements confirm that SKBR3 cells expresshigher levels of Her2-Her2 homodimers than MCF-7 cells.

FIG. 11C shows data from analysis of Her1-Her2 heterodimers and receptorphosphorylation in sections from fixed pellets of breast adenocarcinomacell line, MCF-7, prepared from either non-stimulated cells or cellsstimulated with 40 nM EGF. Two cleaving probes were employed onecomprising anti-Her1 monoclonal antibody (at 5 μg/mL) and the othercomprising anti-Her1 monoclonal antibody (at 10 μg/mL) (both fromLabvision) in order to increase the rate at which molecular tags werereleased. The data show that increases in Her1-Her2 heterodimerexpression due to EGF stimulation is detected in fixed tissue.

FIG. 11D shows data from analysis of Her1-Her2 heterodimers and receptorphosphorylation in sections from fixed pellets of breast adenocarcinomacell line, 22Rv1, prepared from either non-stimulated cells or cellsstimulated with 100 nM EGF. Again, measurements on fixed tissuesdemonstrates the up-regulation of Her1-Her2 dimers and Her receptorphosphorylation in response to treatment with EGF.

FIG. 11E shows data from analysis of Her2-Her3 heterodimers and receptorphosphorylation in sections from fixed pellets of breast adenocarcinomacell line, MCF-7, prepared from either non-stimulated cells or cellsstimulated with 40 nM HRG. In this example, binding reactions andcleavage reactions took place in tubes containing sections, rather thanmicroscope slides. Otherwise, the protocol was essentially the same asthat for detecting the Her1-Her2 dimers. (For example, washing steps arecarried out by centrifugation). The data show that increases inHer2-Her3 heterodimer expression due to HRG stimulation is detected infixed tissue.

FIG. 11F shows data from analysis of Her2-Her3 heterodimers andPI3K-Her3 dimers in sections from fixed pellets of MCF-7 cells eithernon-stimulated or stimulated with 40 nM HRG. The assay design forPI3K-Her3 was essentially as described in FIG. 11A. The above fixationprotocol was followed in both cases, except that neither sample wastreated with antigen retrieval reagents. The data show that Her2-Her3dimers increased with treatment by HRG, but that the amount of PI3K-Her3dimer remained essentially unchanged.

FIG. 11G shows data from analysis of total PI3K, total Her2-Her3 dimer,and total Her3 all relative to amount of tubulin. Tubulin was measuredin a conventional sandwich-type assay employing a cleavage probe and abinding compound with a molecular tag. Tubulin was measured to testprocedures for normalizing dimer measurement against a targetrepresentative of total cell number in a sample, which may be requiredfor measurements on samples with heterogeneous cell types. The data showthat the ratios of PI3K-Her3 and Her2-Her3 to tubulin are qualitativelythe same as the measurements directly on PI3K-Her3 and Her2-Her3.

1. A method of detecting homodimers of membrane-associated analytes on acell membrane, the method comprising the steps of: providing a bindingcompound specific for a membrane-associated analyte forming a homodimer,the binding compound having one or more molecular tags each attachedthereto by a cleavable linkage, the one or more molecular tags eachhaving a different separation characteristic; providing a cleaving probespecific for the membrane-associated analyte, the cleaving probe havinga cleavage-inducing moiety with an effective proximity to the cleavablelinkage, and the cleaving probe and the binding compound being selectedsuch that only one of either the cleaving probe or the bindingcomposition can specifically bind to the same membrane-associatedanalyte at a time; combining the cleaving probe, the binding compound,and the cell membrane such that the cleaving probe and the bindingcompound specifically bind to membrane-associated analytes and such thatcleavable linkages of the binding compound are within the effectiveproximity of the cleavage-inducing moiety whenever a homodimer ispresent and the cleaving probe and the binding compound specificallybind to different membrane-associated analytes thereof, so thatmolecular tags are released; and separating and identifying the releasedmolecular tags to determine the presence or absence or the amount ofhomodimer in the cell membrane.
 2. The method of claim 1 wherein saidmembrane-associated analyte is a cell surface receptor in said cellmembrane and said separation characteristic of said one or moremolecular tags is electrophoretic mobility.
 3. The method of claim 2wherein said step of separating and detecting further includeselectrophoretically separating said released molecular tags in aseparation buffer.
 4. The method of claim 2 wherein saidcleavage-inducing moiety of said cleaving probe is a photosensitizer andwherein said cleaving probe and said binding compound each comprise anantibody binding composition.
 5. The method of claim 4 wherein said cellsurface receptor is selected from the group consisting of epidermalgrowth factor receptors and G-protein coupled receptors.
 6. The methodof claim 5 wherein said cell surface receptor is selected from the groupconsisting of Her1, Her2, Her3, and Her4. 7-14. (canceled)